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Landfill biogas contains certain amounts of H2S that must be removed in order to prevent both equipment corrosion and SO2emissions to the atmosphere when burnt. Anoxic desulfurization has been proven to be an eco-friendly and cost-efficient method to remove H2S from biogas. Nevertheless, and despite all the reported benefits, the potential consumption of methane (CH4) during the anoxic desulfurization of landfill biogas is a factor that has not yet been thoroughly investigated. The present study evaluates the microbial composition and methane assimilation activity of three microbial samples obtained when feeding different nitrate sources, namely nitrified landfill leachate (M1) or chemical nitrate (M2, M3) with 10 days (M2) and 1.5 days (M3) hydraulic residence times. The samples were characterized by the prevalence of sulfide oxidizing bacteria [Thiomicrospira(11.4–25.5 %), Family Rhodobacteraceae(9.9–14.3 %), Sulfurimonas(0.34–17.9 %), Thioclava(0–23.5 %) and Arcobacter(0–11.5 %)], as well as the presence of methane oxidizing bacteria [Halomonas(0.2–16.0 %), Methylophaga(0–0.2 %) and Methylophilacea(0–0.1 %)] and heterotrophic bacteria [Lentimicrobium(0.1–9.7 %) and Roseovarius(0.1–1.2 %)]. The highest CH4assimilation levels were reached under anoxic conditions at 34.0 and 50.1 g CH4m−3h−1using nitrate and nitrite, respectively. The oxygen present in the landfill biogas itself had a detrimental effect on the anoxic bioreactor nitrate removal efficiency. The presence of organic matter in the nitrified influent gave rise to CH4inside the anoxic desulfurization bioreactors, which resulted in the offsetting of the CH4oxidation caused by methane-oxidizing bacteria (MOB).