Antibiotics have the potential to adversely affect the microbial community that is present in biological wastewater treatment processes. The antibiotics that exist in waste streams directly inhibit substrate degradation and also have an influence on the composition of the microbial community. The aim of this study was to evaluate the short-term inhibition impact that various antibiotic combinations had on the syntrophic bacteria, homoacetogenic and methanogenic activities of a microbial community that had been fed with propionate and butyrate as the sole carbon source and VFA mixture (acetate, propionate and butyrate). Acute tests were constructed using on a two way-factorial design, where one factor was the composition of antibiotic mixture and another was the concentration of antibiotics added. In addition, the inhibitory effect of antibiotics was evaluated by monitoring biogas production and the accumulation of individual volatile fatty acids. Specific methanogenic activity batch tests showed a significant (p < 0.05) decrease in the maximum methane production rate in the presence of 1 mg L 1 of antibiotics for the substrate in a VFA mixture and propionate; 1 mg L 1 of ETS, 25 mg L 1 of ET, 10 mg L 1 of ST and ES combination for substrates butyrate. The addition of antibiotics to the batch tests affected the utilization of acetate, propionate and butyrate. This study indicated that antibiotic mixtures have an effect on homoacetogenic bacteria and methanogens, which may exert inhibitory effects on propionate and butyrate-oxidizing syntrophic bacteria, resulting in unfavorable effects on methanogenesis.