Aims: Inhibition behavior of two types of antibiotics including Ampicillin and Gentamicin on specific methanogenic activity of anaerobic biomass has been investigated. Materials and Methods: A total of 18 Specific Methanogenic Activity (SMA) tests were conducted in 120-ml vials containing 40 v/v% substrate, 37 v/v% biomass and 23 v/v% biogas in batch mode for 20-25 days. Produced methane was measured by gas replacement with 2N KOH solution as CO 2 absorbent. Three volatile fatty acids (VFAs) including acetic acid, propionic acid, and butyric acid were used as co-substrate. Results: In the tests with 200, 500 and 1000 mg/l of ampicillin at presence of acetic acid, the cumulative SMA were 66, 101, and 154 ml CH 4 /g VSS.d, those of with propionic acid were 25, 35, and 46 ml CH 4 /g VSS.d, and with butyric acid the values of 198,140, and 245 ml CH 4 /g VSS.d were obtained respectively. In the experiments with 100, 500 and 1000 mg/l of gentamicin the cumulative SMA were 141, 204, and 257 ml CH 4 /g VSS.d for acetic acid as a substrate, 54, 72 and 71 ml CH 4 /g VSS.d for propionic acid, and 139, 74, and 85.5 ml CH 4 /g VSS.d for butyric acid, respectively. Conclusion: At the same concentrations, ampicillin showed more inhibitory effect than gentamicin on anaerobic decomposition of biomass. Within the used VFAs, the inhibitory effects of propionic acid was greater than acetic acid and butyric acid.