After the declaration of war on 10 June 1940, Tembien, now part of the 35 Squadron (III Submarine Group) based out of Messina together with Durbo and Beilul, was assigned to patrol around Malta.

In September 1940, Tembien patrolled off Tobruk, and then participated unsuccessfully in counter-operation against British operation "MB5" (Malta garrison reinforcement).

On 27 November 1940, at 23:24, Tembien sighted three large warships to the south, advancing northwest at an estimated speed of 9 knots.

On 29 June 1941, at 20:41, Tembien sighted a formation of British ships off Ras Azzaz, a hundred miles east of Tobruk.

The group was composed of destroyers HMS Defender and HMAS Waterhen sailing from Tobruk (besieged by the Axis forces) to Alexandria evacuating troops from the 6th Australian Division.

One of these, piloted by Ennio Tarantola, hit HMAS Waterhen on the stern with a 500 kg bomb causing flooding in engine rooms and forcing the crew to abandon the ship.

All three ships were carrying reinforcements and supplies for Malta's garrison and were moving at full speed with the aircraft carrier Ark Royal and destroyers providing distant cover.

In the early hours of 2 August 1941 Tembien sighted British ships while sailing on the surface and tried attacking them unsuccessfully.

After one of the aircraft from Ark Royal spotted the submarine, the rest of the convoy was warned, and HMS Hermione moving at 28 knots maneuvered to ram her.

HMS Hermione sustained only minor damage during collision and was put in for repairs to her bow structures at Gibraltar upon arrival there from her mission on 4 August.