Completed and commissioned in September 1942, she served in World War II, operating in the Solomon Islands, Rabaul, and New Guinea areas.

[4] While she was in the Bismarck Sea northwest of Rabaul on 6 February 1943, an Imperial Japanese Navy Air Service Aichi E13A1 (Allied reporting name "Jake") floatplane mistakenly bombed her, but she suffered only minor damage.

[4] She was 40 nautical miles (74 km; 46 mi) south-southwest of Port Moresby on 14 February 1943 when she sighted an Allied convoy after 16:30 and began a submerged approach at 2.5 knots (4.6 km/h; 2.9 mph) to reach an attack position.

[4] To assist in getting Ro-100 back on an even keel so that her crew could regain control of her before she sank to collapse depth, her commanding officer ordered every available crewman to the forward torpedo room to weigh down her bow.

[4] After the Combined Fleet initiated Operation I-Go — a reinforcement of the 11th Air Fleet base at Rabaul by planes from the aircraft carriers Zuikaku and Zuihō and of the Japanese naval air base on Balalae Island in the Shortland Islands by planes from the aircraft carriers Hiyō and Jun'yō.

[4] She put to sea on 27 May for her third war patrol, again southeast of Guadalcanal, but it also passed quietly, and she returned to Rabaul on 20 June 1943.

[4] While in the Blanche Channel during darkness in early July 1943 conducting a reconnaissance of the American airfield on New Georgia, Ro-100 struck a reef, suffering damage to a fuel tank and two of her bow torpedo tubes.

[4] After reporting her damage, she received orders to pick up stranded Imperial Japanese Navy pilots of the 201st and 204th Naval Air Group at Simbo in the western Solomon Islands and then proceed to Rabaul for repairs.

[4] She was on the surface off Buka Island on 8 August 1943 when an unidentified submarine attacked her; she sighted the wakes of four torpedoes, but all of them missed.

[4] While she was on the surface off Cape St. George on New Ireland that night, an Allied patrol plane surprised her, dropping flares, missing her with two bombs, and strafing her.

[4] While at sea, she was reassigned to Submarine Division 51 on 20 August 1943, but otherwise the patrol was uneventful, and she returned to Rabaul in early September 1943.

[4] After loading a cargo of rubber containers packed with food, she departed Rabaul at 05:00 on 23 November 1943 on an emergency supply run to Buin on Bougainville.