Microorganisms such as bacteria and fungi are developing resistance to the current therapies easily and the presently available antibacterial, antifungal agents and pesticides are certainly very pricey and harmful. So, the necessary shift to use natural antimicrobial extract (Periplaneta americana chitosan) seems to be more effective, economic and safe to the public health. The American cockroach chitosan was dissolved in 1 % acetic acid and diluted to different concentrations then, seeded in 96 well tissue culture plates to evaluate the antibacterial activity by using MTT assay and the microbial growth was calculated by ELISA microplate reader. The American cockroach chitosan showed an equal minimum inhibitory concentration (MIC) antibacterial action against two (Gram-positive) bacterial strains Staphylococcus aureus, and Bacillus subtilis, recording MIC = 2000 µg/ml. Also, chitosan showed MIC antibacterial activity against Escherichia coli and Salmonella typhimurim (Gram-negative) bacteria recording MIC = 1000 µg/ml, and MIC = 2000 µg/ml respectively. While chitosan showed non-antifungal activity against Candida albicans. Natural chitosan could be considered as a determined factor affecting on the biological activities mentioned in this study.