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As part of the naval operations in the Dardanelles Campaign, AE2 made its first attempt to navigate the Dardanelles straits on 24 April, she penetrated 6 nautical miles (11 km) before being forced back with mechanical problems. At 0230 hours on 25 April 1915 under the command of Lieutenant Commander Henry Dacre Stoker, RAN AE2 again attempted to force her way through the straits. At approximately 0430 hours Stoker dived the boat in response to fire from the Turkish guns. By 0600 hours AE2 reached Chanak the narrowest part of the strait and then proceeded to torpedo the Turkish gunboat Peyk I Sevket while simultaneously taking evasive actions to avoid an enemy destroyer. During the evasion Stoker ran the boat aground directly under a Turkish fort, fortunately the fort was unable to lower its guns onto the AE2. After 4 minutes of being exposed on the bank AE2 slid back into deeper waters.

Shortly after grounding a second time the periscope of AE2 was sighted by a Turkish battleship firing over the peninsular at British positions. On sighting the periscope the battleship ceased fire and retreated to a safe position. AE2 continued to make a steady advance toward the Sea of Marmara. At 0830 Stoker decided to rest the boat on the ocean bottom, waiting for the safety of dark. On surfacing he sighted no enemies and proceeded into the Sea of Marmara becoming the first Allied submarine to pass through the Dardanelles Strait.

Stoker signalled his success to his commanders. The commanders of the landings were discussing the possibility of re-embarking the force, the news of AE2's success changed the mood of the conversation and talk of a withdrawal was ended. News of the success of AE2 was spread among Anzac troops to encourage them. AE2 was ordered to Generally run amok, Stoker intended to give the impression that there where multiple submarines in the area. Due to mechanical problems AE2, though making repeated attacks, managed no further hits on Turkish vessels. On 30 April AE2 began to rise uncontrollably and surfaced around 1-nautical-mile (2 km) from the torpedo boat Sultanhisar. Stoker ordered the boat to dive, she went down too deep passing safety levels, then after frantic attempts to resurface the boat she broke surface stern first. Within moments of surfacing she was hit by shellfire, Stoker ordered his crew to abandon ship. All members of the crew survived the attack, though 3 died during the 3 and a half years in captivity. AE2's achievements showed others that the task was possible, within months the Turkish communications line had been badly disrupted.