Abstract: The aim was to study the various antagonistic microorganisms to Fusarium oxysporum. in order to provide a theoretical basis in development of biological preparation. By PDA flat confrontation method and fermentation broth, the competition, hyperparasitism, antibioticaction of penicillium, actinomycete and limewater on Fusarium oxysporum were studied. The result showed: penicillium, actinomycete and limewater had the different Inhibition on the mycelium growth of F. oxysporum, the distance of antagonism were respectively 27.35 mm, 25.70 mm and 16.83 mm, the inhibited rate were 78.36%,75.97% and 47.84%, the inhibited rate of spore germination were respectively 71.97%, 58.23% and 21.49%, in the intersection of penicillium, actinomycete and limewater between Fusarium oxysporum had no obvious hyperparasitism, the conidium of penicillium multiplicated numerously, occupied majority of the space. Penicillium had apparent effect on Fusarium oxysporum in the competition and antibioticaction, had the best inhibition.