The present study is related to new green and low cost technical method for wastewater treatment using chitosan as natural material. Chitosan was prepared from chitin of the seafood's shell (Procambrus clarkia). Preparation of chitosan was carried out by a chemical process involving demineralization, deproteinization and deacetylation. Chitosan is largely known for its activity against a wide range of microorganisms. Antibacterial effect of different doses of chitosan (0.09, 0.2, 0.4, 0.6 and 0.8 g) on Escherichia coli (E. coli) bacteria in the secondary treated wastewater samples was studied. The secondary treated wastewater was collected from Zenein wastewater treatment plant. Also the effectiveness of chitosan on Enterococcus faecalis, Bacillus subtillus, Staphylococcu saureus (gram +ve bacteria), Escherichia coli, Enterobacter aerogenes (gram –ve) was evaluated by the reduction in total bacterial count of each strain in synthetic media. The study was extended to measure the antibacterial activity (inhibition zone) of chitosan on E. coli by the poured plate method. The results indicated that the optimum conditions at which 99.98% removal for all types of bacteria were 0.6 g chitosan, contact time 30 min and shaking 250 rpm. According to the obtained results, the most acceptable antimicrobial mechanism is found to include the presence of charged groups in the polymer backbone and their ionic interactions with bacterial wall constituents. It could be concluded that chitosan could be used for disinfection of the secondary treated wastewater.