Sitafloxacin is one of the newer generation fluoroquinolones. Considering the ever-changing antimicrobial resistance, it is necessary to monitor the activities of sitafloxacin against recent pathogenic isolates. Therefore, we determined the minimum inhibitory concentrations (MICs) of sitafloxacin and comparators by broth microdilution or agar dilution method against 1101 clinical isolates collected from 2017 to 2019 in 31 hospitals across China. Sitafloxacin was highly active against gram-positive isolates evidenced by the MICs required to inhibit the growth of 50%/90% isolates (MIC): ≤ 0.03/0.25, ≤ 0.03/0.125, ≤ 0.03/2, 0.125/0.25, 0.25/2, and 0.125/0.125 mg/L for methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-susceptible coagulase-negative Staphylococcus (MSCNS), methicillin-resistant S. aureus (MRSA), methicillin-resistant CNS, Enterococcus faecalis, and Streptococcus pneumoniae, respectively. Sitafloxacin inhibited 82.8% of the MRSA strains and 97.5% of MRCNS strains. Sitafloxacin was also potent against ciprofloxacin-susceptible Escherichia coli (MIC: ≤ 0.03/0.06 mg/L) and Klebsiella pneumoniae (MIC: ≤ 0.03/0.125 mg/L), non-ESBL-producing E. coli (MIC: ≤ 0.03/1 mg/L) and K. pneumoniae (MIC: ≤ 0.03/0.5 mg/L), Haemophilus influenzae (MIC: ≤0.015/0.06 mg/L), Haemophilus parainfluenzae (MIC: 0.125/0.5 mg/L), Moraxella catarrhalis (MIC: ≤ 0.015/≤ 0.015 mg/L), Bacteroides fragilis (MIC: 0.06/2 mg/L), Peptostreptococcus (MIC: 0.125/4 mg/L), and Mycoplasma pneumoniae (≤ 0.03/≤ 0.03 mg/L). However, sitafloxacin was less active for Enterococcus faecium, ciprofloxacin-resistant and/or ESBL-producing E. coli, and K. pneumoniae strains. Sitafloxacin was superior or comparable to most of the comparators in activities against the abovementioned isolates, so sitafloxacin is still highly active against most of the clinical isolates in hospitals across China, proving its utility in treatment of the abovementioned susceptible strains.