The susceptibilities of clinical isolates to fluoroquinolones and other antimicrobial agents were surveyed to obtain an accurate understanding of trends in incidence and antimicrobial resistance. The samples were collected from across Japan, biennially or triennially, between 1994 and 2016 and a defined level of resistance to fluoroquinolone was determined. Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae exhibited stable and high rates of susceptibility to fluoroquinolones over the period examined. For methicillin-resistant Staphylococcus aureus the rate of resistance to levofloxacin and ciprofloxacin was 81.3e93.5% and 83.2e94.2%, respectively, which was markedly higher than that of methicillin-susceptible S. aureus, while sitafloxacin-resistant methicillin-susceptible and methicillin-resistant S. aureus were isolated at 0.3e0.7% and 16.9e36.5%, respectively. The rate of levofloxacin or ciprofloxacin-resistant Escherichia coli increased from around 2e3% between 1994 and 1998 to around 35% in 2016, but the rate of fluoroquinolonesusceptible Klebsiella pneumoniae stayed high at over 94.6% during the study period. Although no fluoroquinolone-resistance in clinical isolates of Salmonella spp. was detected from 1994 to 2002, the resistance rate increased slightly after 2004 and reached to 1.9%e4.7% in 2016. The rate of fluoroquinolone-susceptible Pseudomonas aeruginosa isolated from urinary tract and respiratory tract infections improved during the period examined from 41.8e67.0% to 91.2e94.2%, and from 78.9-88.5% to 90.1e94.6%, respectively. Against Acinetobacter spp., the susceptibility rate of fluoroquinolones was almost constant at around 90%, but one multidrug-resistant isolate was detected in 2013. Overall, the susceptibility to fluoroquinolones was maintained over 20 years against tested bacteria except for MRSA and E. coli.