Objective To investigate the bacterial resistance profile of clinical isolates collected in the hospitals across Chuzhou in 2016. Methods Antimicrobial susceptibility testing was carried out by Kirby-Bauer method. The data were analyzed using WHONET 5.6 software according to CLSI 2015 breakpoints. Results A total of 5 465 clinical isolates were collected during 2016, of which gram positive organisms and gram negative organisms accounted for 25.9％ (1 416/5 465) and 74.1％ (4 049/5 465), respectively. Prevalence of MRSA was 37.6％ among S. aureus and the prevalence of MRCNS was 78.1％ in CNS. All Staphylococcus, E. faecalis and E. faecium isolates were sensitive to vancomycin and linezolid. The prevalence of extended spectrum-lactamases (ESBLs) positive strains was 51.2％ in E. coli, 23.4％ in Klebsiella spp. (K. pneumoniae and K. oxytoca), and 23.6％ in P. mirabilis isolates, respectively. The Enterobacteriaceae strains were highly sensitive to carbapenems. The percentage of the P. aeruginosa isolates resistant to the antimicrobials tested was lower than 30％. The percentage of the Acinetobacter strains resistant to meropenem and imipenem was 65.6％ and 67.4％, respectively. Conclusions The situation of antibiotic resistance is still very serious, especially multi-drug or pan-drug resistant strains, which is of great concern.