Malathion kills insects by preventing their nervous system from working properly. When healthy nerves send signals to each other, a special chemical messenger travels from one nerve to another to continue the message. The nerve signal stops when an enzyme is released into the space between the nerves. Malathion binds to the enzyme and prevents the nerve signal from stopping. This causes the nerves to signal each other without stopping. The constant nerve signals make it so the insects can't move or breathe normally and they die.

People, pets and other animals can be affected the same way as insects if they are exposed to enough malathion. About the same amount of malathion will be taken into the body whether you breathe it in or you swallow it. Malathion is also readily taken into the body through skin, though the amount absorbed will depend on where the exposure occurs on the body. Malathion can become more toxic if it has been sitting for a long time, especially in a hot place.