Methane fermentation has been widely used to dispose sewage sludge at wastewater treatment plants (WWTPs), due to production of renewable energy in form of biogas. Antibiotics present in wastewater might accumulate in a sewage sludge. The aim of the present study was to investigate the impact of three antibiotics from different classes in three different doses on methane production from sewage sludge. For this purpose, metronidazole (MET), amoxicillin (AMO) and ciprofloxacin (CIP) were individually added to anaerobic reactors with sewage sludge collected from municipal WWTP. The antibiotics highest concentration (1024 mg/kg of AMO; 512 mg/kg of MET and CIP) lowered methane production and methane content in biogas. MET exerted the most marked effect and lowered methane production to 36.8 ± 3.7 mL CH4/kg volatile solids (VS). Tested antibiotics probably inhibited methanogenic Archaea, which resulted in volatile fatty acids (VFAs) accumulation. Addition of MET resulted in accumulation of many kinds of VFAs with the highest concentration of acetic acid (17.52 ± 1.85 g/L). Addition of AMO resulted in accumulation of butyric acid only (253.00 ± 15.89 g/L). However, addition of CIP resulted in accumulation mainly acetic acid (7.58 ± 0.82 g/L) and isovaleric acid (2.01 ± 0.41 g/L). Next, synergistic effect of these antibiotics in a low concentration of 16 mg/kg of AMO, 8 mg/kg of MET, and 2 mg/kg of CIP was measured in semi‐continuous conditions. These low concentrations of antibiotics individually slightly influenced methane fermentation, however synergistic effect of them caused inhibition of methane production and accumulation of VFAs. This article is protected by copyright. All rights reserved