Methanotrophs are of great significance for the abatement of methane emission from anoxic environments. Antibiotics are ubiquitous in the environment and can affect microbial activity and community density and composition. However, information about the effect of antibiotics on methanotrophs is still lacking. The current study explored the influences of sulfonamides and tetracyclines on methane oxidation potential (MOP) and methanotrophic density and community structure in freshwater sediment microcosms. The addition of both sulfanilamide (SA) and oxytetracycline (OTC) could increase MOP and particulate methane monooxygenase subunit A (pmoA) gene density but decrease the number of pmoA transcripts. Both SA and OTC could also have impacts on sediment methanotrophic community structure. The antibiotic effects on MOP and methanotrophs were found to depend on the dosage and type of antibiotics. This work could provide some new insights towards the links between methane oxidation and antibiotics.