During World War II, the harbour and its facilities accommodated scores of Australian and Allied naval vessels on active service. Battleships, troop transports, hospital ships and support vessels, including many passenger ships, were seconded into the war effort.

Among famous visitors to pass through Fremantle during the Second World War were the massive liners, then war service troop carriers, QUEEN ELIZABETH and QUEEN MARY. However, because of their size neither was able to take up an Inner Harbour berth, and instead anchored in Gage Roads.

Some other well-known ships used as troop transports were P&O and Orient Liners STRATHAIRD, STRATHNAVER, ORFORD, ORION, ORCADES and OTRANTO.

Interstate passenger vessels such as the KANIMBLA, MANOORA and WESTRALIA were also requisitioned for war service.

Boom defences were also placed in operation in December 1940 as a security measure to control shipping movements, and anti aircraft installations were established around the harbour to guard against any enemy air attacks.

Fremantle had a major role as the largest submarine base in the Southern Hemisphere during World War II. The first United States submarines arrived at Fremantle in 1942, and over the next three years, the port accommodated more than 170 submarines from the U.S., British and Dutch navies.

The United States Navy established a submarine repair facility on North Quay in 1943.

The submarine base was a valuable source of employment to Fremantle during the war, but shortly afterward much of the wartime infrastructure reverted to its pre-war use.

Once the shipping lanes were safe after the war, the Port of Fremantle continued its busy life with the arrival of merchant ships and ocean liners. Their trade was no longer in troops but in immigrants arriving from Britain and war-torn Europe looking for a new life.