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Introduction: Antibiotics have the potential to adversely affect the microbial community. For anaerobic digestion, a sufficient methanogenic population needs to be preserved in the system. The main aim of this study was determination of inhibitory concentration of oxytetracycline on methanogenic bacteria. Methods: A 120 mL jacketed bioreactor with a 90 mL working volume was inoculated granular sludge from an anaerobic digester, substrate and different concentration of oxytetracycline with 10 days cycles and intermittent mixing. The reactor was operated at 35 ± 2 ° C. The inhibitory effect of antibiotic was evaluated by monitoring biogas production. Results: Based on the findings from each batch, complete inhibitory concentration of oxy tetracycline was in concentration of 800 mg L-1. Significant relation was seen between inoculated antibiotic concentrations and methane production (r=-0.86). Conclusion: The addition of antibiotics to the biomass affected the utilization of fatty acids, resulting in unfavorable effects on methanogenesis. Thus, overusing of antibiotics can adverse effects of intestinal flora.