Although zinc oxide nanoparticles are known as an effective antimicrobial agent, the mechanism for its antifungal activity has been assessed by only few studies. In this study, antifungal activity mechanism for ZnO via the change of physiology was explored. It was showed that the activities of SOD and CAT and the MDA content were increased significantly. The possible mechanisms were obtained that zinc oxide directly acts on the mycelium of penicillium to produce oxidative stress and destroy intracellular physiological balance. RNA-seq was then used to verify the conclusion obtained before, and obtained results suggested that the antifungal mechanism is attributed to oxidative stress and changes in membrane function.