This study analyzed the utility of ammonium chloride (NH4Cl) as a nitrogen source for methanotroph communities. When cultured in nitrate mineral salt (NMS) medium, the methanotroph community we identified four families, seven genera, and 16 type I and type II species of methanotrophs. Among species in the Methylobacter genus, Methylobacter marinus could be actively cultured in NMS medium without NaCl addition. Following the addition of 25 mM NH4Cl, the numbers of the type I genera Methylomonas, Methylococcus, and Methylobacter were increased, whereas the numbers of the type II genera Methylocystis and Methylosinus were decreased after 5 days. In methanotroph communities, certain concentrations of NH4Cl affected methane consumption and growth of methanotrophs at the community level. NH4Cl caused a considerable decrease in the methane consumption rate and the expression of soluble methane monooxygenases (sMMOs) but did not inhibit the growth of Methylomonas methanica expressing sMMO. These results could be attributed to competitive antagonism of MMOs due to their direct involvement in ammonia oxidation.