The study evaluated the short-term inhibition impact of three antimicrobials, sulfamethoxazole, erythromycin and tetracycline, on the methanogenic activity of acclimated biomass fed with acetate. Batch reactors were inoculated each with a different antimicrobial concentration in the range of 1-1000 mg/L and they were operated during 6 days. Organic substrate removal was monitored by both soluble COD and acetate measurements, together with daily measurements of biogas and methane generation. While acetate was almost fully removed in all experiments, methane generation exhibited a significant drop with increasing antimicrobial doses. Almost complete methane inhibition was observed for antimicrobial doses above 500 mg/L. Together with adverse impact on process kinetics in the early phases of the experiments, the final acute impact of antimicrobials was on process stoichiometry, preventing complete utilization of acetate removed in metabolic reactions. The observed effect was found compatible with uncompetitive inhibition, which similarly exerts a binding impact on substrate-enzyme complex. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.