This study showed the effect of amoxicillin (AMO), and oxytetracycline (OXY) at a concentration of 512 µg mL−1, and sulfamethoxazole (SMX), and metronidazole (MET) at a concentration of 1024 µg mL−1 on the efficiency of anaerobic digestion (AD) of sewage sludge (SS) and cattle slurry (CS). The production of biogas and methane (CH4) content, and the concentration of volatile fatty acids (VFAs) was analyzed in this study. Other determinations included the concentration of the mcrA gene, which catalyzes the methanogenesis, and analysis of MSC and MST gene concentration, characteristic of the families Methanosarcinaceae and Methanosaetaceae (Archaea). Both substrates differed in the composition of microbial communities, and in the sensitivity of these microorganisms to particular antimicrobial substances. Metronidazole inhibited SS fermentation to the greatest extent (sixfold decrease in biogas production and over 50% decrease in the content of CH4). The lowest concentrations of the mcrA gene (106 gD−1) were observed in CS and SS digestates with MET. A decline in the number of copies of the MSC and MST genes was noted in most of the digestate samples with antimicrobials supplementation. Due to selective pressure, antimicrobials led to a considerably lowered efficiency of the AD process and induced changes in the structure of methanogenic biodiversity.