Technical Abstract: Pythium ultimum Trow is a plant pathogen that causes significant yield losses on many economically important crops. Chemical seed treatment has been used for disease control. In searching for alternatives, the venom alkaloids from red imported fire ant were tested against P. ultimum in vitro and to control cucumber damping-off in the greenhouse. Piperidine and piperideine alkaloids of the red imported fire ant significantly inhibited the mycelial growth of P. ultimum. Piperidine alkaloids were stable at both room and elevated temperatures. The inhibitory activity positively corresponded to the concentrations of piperidine alkaloids in the medium, and the EC50 = 17.00 µg mL-1. Germination of sporangia of P. ultimum negatively corresponded to the concentrations of piperidine alkaloids in the medium, and the EC50 = 12.25 µg mL-1. Drenching treatment significantly improved the seedling emergence and seedling height of cucumber in greenhouse. This is the first report using piperidine and piperideine alkaloids from the red imported fire ant to control damping-off disease caused by P. ultimum. These findings may lead to the development of a new group of fungicides.