Bio-oxidation of methane in a landfill environment is important for mitigating global methane emissions into the atmosphere. In the present study, the characteristics of methane bio-oxidation and methane-oxidizing microorganisms were studied by enrichment cultivation using fresh and aged leachate (collected from Qizishan Landfill, Suzhou, China). Both aerobic and anaerobic methane oxidation were detected, methane oxidation capacities of the culture liquid were 5.24–7.26 μmol/mL/d under aerobic conditions and 4.41-3.70 μmol/mL/d under anaerobic conditions. The stoichiometry of anaerobic oxidation of methane (AOM) showed the complexity of AOM mechanisms in the leachate culture, with the types of sulfate-dependent, denitrification-dependent and iron-dependent AOM. The 16S rRNA gene sequence analysis and SEM analysis results showed that the genus Methylocystis was the dominant bacteria in aerobic cultures (relative abundance 35.96–78.37%). Genus Moheibacter (41.38%) and Cupriavidus (43.08%) were the most dominant taxa in anaerobic cultures, with aerobic methanotrophs Methylocaldum and Methylocystis in low abundance, and no anaerobic methane-oxidizing archaea (ANME) was found. Further research is needed to confirm whether aerobic methanotrophs can oxidize methane under anaerobic conditions.