Abstract: Cell wall of zygomycetes fungus is an alternative source for chitosan production. In this study chitosan was extracted from cell wall of filamentous fungus Rhizopus oryzae and its antimicrobial properties was studied against three typical human pathogenic microorganisms, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. The viability of these bacteria reduced by more than 60%, when 200 ppm of the fungal chitosan was present in the solution. However, the Minimum Bactericidal Concentration (MBC) of the fungal chitosan was 300, 500 and 700 ppm for S. aureus, E. coli and K. pneumoniae, respectively. The antimicrobial activity of fungal chitosan was lower than that of crustacean shells chitosan, which had MBC of less than 100 ppm for the above mentioned bacteria. Furthermore, fungal chitosan similar to crustacean shells chitosan exhibited better inhibitory effects against gram-positive compared to gram-negative bacteria. The possible mechanism for antimicrobial activity of fungal chitosan could be the disruption of the outer membrane of cells but not preventing the nutrients from entering into the cell.