Robert Morley and Katharine Hepburn play Samuel and Rose Sayer, brother and sister British missionaries in a village in German East Africa in 1914. Their mail and supplies are delivered by the rough-and-ready Canadian boat captain Charlie Allnut (Humphrey Bogart) of the African Queen, whose coarse behavior they tolerate in a rather stiff manner. When Charlie warns them that German troops will soon invade, the Sayers choose to stay on, only to witness the Germans burning down the local village and herding the villagers away to serve in the war. When Samuel protests, he is beaten by a German soldier. After the Germans leave, Samuel becomes delirious and dies.

Soon afterward, Charlie returns, and after helping Rose bury her brother, they set off in the African Queen. Charlie tells Rose that the Germans have a gunboat, the Empress Louisa, which patrols a large lake downriver, effectively blocking any British counterattacks. Rose still wants Charlie to pilot the boat down to the lake. Charlie points out that navigating the river would be suicidal: to reach the lake they would have to pass a German fort and negotiate several dangerous rapids. But Rose is insistent and eventually persuades him to go along with the plan. Charlie is furious when the teetotaler Rose throws away all of his gin, but she insists that he needs to be sober for the task at hand. Charlie hoped after passing the first obstacle that Rose would be discouraged, but she is confident they can handle what is yet to come, and argues that Charlie promised to go all the way.

During their journey down the river, Charlie, Rose and the African Queen encounter many obstacles, including a German fortress perched on a hilltop near the river (with the drafted native villagers shooting at them) and three sets of rapids. The first set of rapids is rather easy; they get through with minimal flooding in the boat. But Rose and Charlie have to duck down when they pass the fortress and the soldiers begin shooting at them, blowing two bullet holes in the top of the boiler and causing one of the steam pressure hoses to disconnect from the boiler, which in turn, causes the boat's engine to stop running. Luckily, Charlie manages to reattach the hose to the boiler just as they are about to enter the second set of rapids. The boat rolls and pitches crazily as it goes down the rapids, leading to more severe flooding in the boat and also collapsing the stern canopy. Later on, the couple decide to take a pit stop to gather more fuel and drain the boat. Back on the river, Charlie and Rose watch crocodiles frolick on the nearby river bank when the third set of rapids comes up. This time, there is a loud metallic clattering noise as the boat goes over the falls. Once again, the couple dock on the river bank to check for damage. When Charlie dives under the boat, he finds the propeller shaft bent sideways and a blade missing from the propeller. Luckily, with some expert skill using suggestions from Rose, Charlie manages to straighten the shaft and weld a new blade on to the propeller, and they are off again.

All appears lost when Charlie and Rose "lose the channel" and the African Queen becomes mired in the mud amid dense reeds. First, they try to tow the boat through the muck, only to have Charlie come out of the water covered with leeches. In the end, Rose and Charlie go to sleep. As they sleep, exhausted and beaten, heavy rains raise the river's level and float the Queen off of the mud and into the lake which, it turns out, is just a short distance from their location. Once on the lake, they narrowly avoid being spotted by the Louisa.

Rose comes up with a plan to convert the Queen into a torpedo boat and sink the Louisa. That night, they set about converting some oxygen cylinders into torpedoes using gelatin explosives and improvised detonators that use nails as the firing pins for rifle cartridges. They then attach the torpedoes through the bow of the Queen. At the height of a storm they push the Queen out onto the lake, intending to set it on a collision course with the Louisa. Unfortunately, the holes in the bow in which the torpedoes were pushed through are not sealed, allowing water to pour into the boat, causing it to sink lower and eventually the Queen tips over. Charlie is captured and taken aboard the Louisa, and after being questioned, Rose is captured and Charlie hollers her name, then pretends not to know her. The captain questions her, and Rose says they planned to sink the German boat and encourages Charlie to describe his torpedoes. The captain sentences them to be executed as spies; Charlie asks the German captain to marry them before executing them. After a brief marriage ceremony, the Germans prepare to hang them, when there is a sudden explosion and the Louisa starts to sink. The Louisa has struck the overturned hull of the African Queen and detonated the torpedoes. Rose's plan has worked, if a little belatedly, and the newly-married couple swim to safety.

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