To observe the antibacterial activity of moxalactam against Enterobacteriaceae bacteria and anaerobic bacteria in vitro, and to compare with other antibacterial drugs, for providing experimental basis for the clinical application of moxalactam.Minimum inhibitory concentrations (MICs) of moxalactam and other antibacterial agents against 491 Enterobacteriaceae spp. and anaerobic spp.collecting from clinical settings were determined by agar dilution methods and E-test strips according to the Clinical and Laboratory Standards Institute (CLSI)(2014).Moxalactam showed great antibacterial activity to Enterobacteriaceae spp., including ESBLs-producing Escherichia coli, Klebsiella pneumoniae, and Proteus spp., with the MIC(50), MIC(90), and susceptibility rates of 0.25-4 mg/L, 0.5-8 mg/L, and >90%, respectively. The susceptibility rates of Enterobacteriaceae with ESBLs-producing or non-ESBLs-producing to imipenem and meropenem were both higher than 90%. The susceptibility rates of ESBLs-producing Escherichia coli, Klebsiella pneumoniae, and Proteus spp.to piperacillin/tazobactam and cefoperazone/sulbactam were 90%, 68%, 53% and 76%, 66%, 76.6%, respectively, while the susceptibility rates of non-ESBLs-producing Escherichia coli, Klebsiella pneumoniae, and Proteus spp.were all more than 95%. The susceptibility rates of Enterobacter spp. and other Enterobacter to piperacillin/tazobactam were 80%, 80%and that to cefoperazone/sulbactam were 80%, 76.7%, respectively.The MICs range of moxalactam on anaerobic spp.was from ≤0.064 to >256 mg/L, while MIC(50) was 2 mg/L and MIC(90) was 64 mg/L.Moxalactam showed well activity against ESBLs-producing and non-ESBLs-producing Enterobacteriaceae and anaerobia.