The presence of antibiotics in drinking water and wastewater has not been widely studied because the sanitary engineering sector mainly focuses on the removal of organic matter and nutrients. There is a lack of environmental regulations for pollutants like antibiotics. Batch tests analyse biodegradability to measure the anaerobic degradation potential of the substrate, or they can be used as toxicity tests. Oxytetracycline, florfenicol (FLO), ceftiofur (CEF) and penicillin G (PEN), commonly used in Colombia for the treatment of livestock diseases, were added in different concentrations to anaerobic sludge contained in serological glass bottles. The production of methane stored in the empty spaces of the bottles was monitored in order to determine the effect of the aforementioned antibiotics on the anaerobic process. It was found that CEF did not have any inhibitory effect on methanogenic activity, while PEN showed inhibition at all concentrations evaluated.