To better study the inhibition effect of three strains of antagonistic bacteria against Pestalotiops photiniae C16-4 ,the using of scanning electron microscopy (SEM) is a good way. This paper studies three kinds of methods (cake sample, insert sample and not fixed sample) for preparation of specimen observing fungus. Through the inhibition of fungus cultivation, we observed the ultrastructure of fungus. Insert sample without fixed was a short, simple, rapid method. Insert sample was more practical for conventional observation and identification of fungi and provided a reference for SEM of other microorganisms. This preparation procedure could be most useful for the routine examination and identification of the fungus. The results show that the changes of hyphae structure after treatment of different antagonistic bacteria inhibition can be seen, which mainly showing adhesion between mycelium, uneven thickness, partly sunken, or fracture, which giving full play to its bacteriostatic action.