Aims: The purpose of this study was to survey the antibiotics effects of oxytetracycline, tylosin, and amoxicillin on anerobic wastewater treatment process. Materials and Methods: To evaluate the inhibitory antibiotics amoxicillin, tetracycline, and tylosin on biomass activity, specific methanogenic activity (SMA) using anerobic biomass batch; into 120 ml vials: 30 ml biomass and 70 ml substrate including volatile fatty acids, mainly acetic acid and various concentrations of antibiotics were added. Methane gas production replacement through solution of KOH (2 N) as an absorber of CO 2 and bromine thymol blue as indicator was measured. Each batch was tested for 10 days. Results: Based on the findings, inhibitory concentration of oxytetracycline, amoxicillin, and tylosin were 8000, 9000, and 9000 mg/L, respectively. Conclusions: This study showed that with increasing concentrations of antibiotics, the produced biogas volume from biomass per unit weight is decreased. COD removal was 42-82 % due to long retention time and adsorption to flocks.