Following further training off Colón, Panama, the submarine returned to New London, Connecticut, before holding her acceptance trials and undergoing a post-shakedown overhaul at the Portsmouth Navy Yard in Kittery, Maine.

After conducting live-fire trials on the effectiveness of depth charges, the first of their kind in the U.S. Navy, Tambor reported in May 1941 to the Submarine Force, Pacific Fleet, and the command of Rear Admiral Thomas Withers (COMSUBPAC).

Tambor began her first war patrol[9] on 15 March 1942 when she stood out of Pearl Harbor to reconnoiter the areas around Wake Island, Truk, New Ireland, New Britain, and Rabaul.

The group of six submarines sailed for Midway Island on 21 May 1942 to begin patrolling a 150 miles (240 km) circle in anticipation of the invasion fleet intelligence had reported was en route there.

At 07:15 on 4 June 90 minutes after first reported contact, COMSUBPAC, Admiral Robert H. English, informed his submarines, waiting until after 11:00 to order them to close.

At 0258, Murphy turned due west, hoping to put them between him and the moon so their silhouettes would make them easier to identify.

After losing and regaining sight of the cruisers in the dark, Murphy made an educated guess, turning left again, and re-sighted the ships.

When there was no attack, the sub was brought to periscope depth, where damage to the cruiser Mogami's bow was easily visible.

[19] When Tambor had sailed across in front of the line of cruisers in an effort to see them more clearly, the lead ship had sighted the sub and ordered a change of course.

Mogami had turned a bit late and had collided with Mikuma, sailing just in front of her, and 40 feet of her bow was compressed and pushed nearly perpendicular to the rest of the ship.

The next day, based on Tambor's report, and following the oil slick, dive bombers found the two damaged cruisers and their destroyer escorts and were able to sink Mikuma—the largest Japanese non-carrier sunk to that point in the war.

Since surface pursuit would have been suicide, as the cruisers could have easily destroyed Tambor with their guns, the sub was forced to submerge.

Murphy was promptly relieved of command, for timidity in the face of the enemy in light of Tambor's failure to close with and attack the enemy, or at least identify and properly shadow the Japanese cruiser force so aircraft could be vectored to the location (a regular task for both Japanese and American submarines and the primary purpose for which Tambor was there at all).

Murphy's lack of aggressiveness had hampered Spruance's intelligence of the battle and had played an important role in allowing Suzuya, Kumano and Mogami to escape almost certain destruction from air attack.

[23][24] Her next patrol (now in the hands of Stephen H. Ambruster) began on 24 July 1942 at Pearl Harbor, ending on 19 September at Fremantle, Australia.

On 7 August near Wotje Atoll, she sank the converted net tender Shofaka with one torpedo which broke her in half.

[25] Tambor sailed for Hainan Strait on 12 October 1942 and (in part due to torpedo shortages) laid mines.

[27] Tambor sailed from Fremantle on 18 February 1943 to carry out a special mission in the Philippine Islands, in support of "MacArthur's Guerrillas".

{This was the Bugen Maru, which was damaged}[27] Seven days later, she scored one hit on a freighter out of three torpedoes fired and believed it sank;[28] it was not confirmed.

On 27 July the Teiken Maru was sunk by a mine laid by the Tambor[30] On 3 August, she sighted five cargomen and a destroyer in Palawan Passage.

Tambor opened fire with her aft 20-millimeter gun and turned hard to port causing the escort to pass 20 yards (20 m) astern.

[31] After refit at Pearl Harbor, Tambor put to sea on 9 April 1944 en route to the Mariana Islands.

In a submerged attack, Tambor fired four torpedoes at a cargo ship and heard two explosions, then went deep, taking 50 depth charges from the escorts.

The submarine (now in the hands of William J. Germershausen)[33] conducted her next patrol in the waters off southern Hokkaidō and the Kuril Islands from 16 July to 23 August 1944.

{possibly the escort Kunashiri reported to have been damaged by "unknown cause"} On 13 August, Tambor made a surface attack against a cargo ship and then photographed Toei Maru (2,300 tons)[34] as she lowered two lifeboats and sank in 20 minutes.

The submarine returned to Saipan from 8 to 10 November, then resumed her patrol, now one of six members of the wolfpack "Burt's Brooms" (named for Thomas B. Klakring, commanding SubDiv 101).

At 06:10, the submarine's commander decided to battle on the surface with his deck guns; the escort fired back, and a Tambor crewmen was severely wounded.

{The sunken vessel was the guardship "Takashiro Maru"}[27]Tambor ended her last war patrol at Pearl Harbor on 30 November 1944, to be retired from combat.

Upon her arrival, Tambor began training operations with Navy patrol aircraft under Fleet Air Wing 6.

She spent five weeks in drydock for a routine overhaul, being covered in 7,000 pounds (3,175 kg) of paint, including six coats on her anchor chain.