Urinary tract infections (UTIs) and the antibiotic resistance of pathogenic bacteria pose severe threats to public health in the current healthcare environment. The purpose of this study was to assess the frequency distribution of bacterial pathogens causing UTIs as well as the characteristics of antibiotic susceptibility and resistance. The retrospective study was conducted on 32,391 samples of midstream urine culture from January 1, 2020, to December 31, 2022, in Jiaxing. Bacteria were cultivated on blood agar and identified using MALDI-TOF, and their susceptibility to different antibiotics was assessed using the Kirby-Bauer disk diffusion method and drug sensitivity reaction cards. The SPSS 22 software was used for data analysis. Bivariate logistic regression was used to analyze the risk factors for multidrug resistance. The total number of positive growth samples was 5378 (16.6%), including 3206 females (59.6%) and 2172 males (40.4%). The four most common urinary pathogens were Escherichia coli (39.2%), Enterococcus faecalis (12.4%), Klebsiella pneumoniae (7.6%), and Enterococcus faecium (7.6%). As far as antibiotic resistance was concerned, Escherichia coli had a greater than 50% resistance rate to ampicillin (76.1%), ciprofloxacin (58.6%), and levofloxacin (51.2%). The multidrug resistance rate was high (41.8%). Low levels of resistance were seen to ertapenem (0.1%), imipenem (0.7%), meropenem (0.7%), piperacillin/tazobactam (0.7%), and nitrofurantoin (1.8%). Klebsiella pneumoniae was highly sensitive to ertapenem (100%). The resistance rates to nitrofurantoin, ceftriaxone, and ciprofloxacin were 37.4%, 37.1%, and 35.1%, respectively. Up to 41% of Escherichia coli strains and 26% of Klebsiella pneumoniae strains produced extended-spectrum lactamases (ESBL). Two species of enterococci were highly sensitive to tigecycline and linezolid (100%), and a small number of norvancomycin-resistant strains (0.2%/two strains) were found. Escherichia coli and Enterococcus faecium were the most common urinary pathogens in this study. The isolated pathogens showed different sensitivity patterns. Antibiotics should be selected reasonably according to the sensitivity mode of pathogenic bacteria to effectively treat and prevent urinary tract infections.