Knowing recent drug-resistant bacteria trends is important for proper antibacterial drug use to improve the prognosis of patients with infectious diseases and for public health. Because multiple quinolone antibacterial agents are simultaneously adopted in hospitals in Japan, we examined whether minimum inhibitory concentrations （MICs） against Haemophilus influenzae differ among quinolones. We determined MICs of six different quinolone antibacterial agents and performed molecular genetic analysis. We investigated β-lactamase-producing and β-lactamase-negative ampicillin-resistant（BLNAR）H. influenzae using the nitrocefin method in parallel. Overall, 144 clinical H. influenzae strains isolated at the Showa University Hospital between 2012 and 2017 were subjected to MIC determination for penicillin/quinolone antibacterial agents using the Clinical and Laboratory Standards Institute broth microdilution method. Amino acid mutations in the quinolone resistance-determining regions were analyzed in the isolates showing an MIC value ≥ 0.25µg/ml of quinolone antibacterial agents. BLNAR isolates increased from 2016 onward. Among quinolone antibacterial agents, all isolates remained susceptible to sitafloxacin. However, for moxifloxacin（MFLX）, strains with an MIC value＝0.5µg/ml were detected every year since 2013 except in 2015. Amino acid mutations were investigated in 17 isolates （11.8％） with MFLX MIC value≥ 0.25µg/ml and confirmed in 11 isolates （7.6％）, of which 9 contained GyrA mutations. The results demonstrated that MFLX was useful for predicting the presence of amino acid mutations and 0.25 was an appropriate MIC threshold for this purpose. This screening procedure may be effective for reducing the inappropriate use of quinolones and controlling the emergence of drug-resistant H. influenzae.