At present, there is a distinct lack of understanding to the effect of antibiotic contamination of a substrate on two-stage anaerobic digestion (AD) processes. The aim of this study was to investigate how oxytetracycline (OTC) influences the structure of the microbial community and the production of methane in two-stage anaerobic cattle manure digesters. Ion Torrent sequencing and PCR-DGGE analysis were used to detect microbial community changed in response to the addition of oxytetracycline. Illumina sequencing was also performed to detect antibiotic resistance genes (ARGs). Digestion studies utilized samples of manure produced by cattle which had been therapeutically treated using a standard OTC solution of 50 mL per 20 mg kg(-1) cattle weight. According to the results of the PCR-DGGE and Ion Torrent sequencing, the bacterial diversity of acidogenic digesters was higher than those of single-stage digesters during the digestion period. An assessment of single-stage and two-stage AD data highlighted that Methanosarcina and syntrophic acetate-oxidizing bacteria are critical to the production of biogas. The overall results indicated that changes in the structure of a microbial community lead to changes in biodegradation capacity of antibiotics and occurrence of ARGs. (C) 2016 Elsevier B.V. All rights reserved.