Objective: To investigate the pathogen composition and drug susceptibility of patients with nosocomial infection. Methods: 104 patients with acquired infection admitted to our hospital from September 2023 to June 2024 were selected as the study objects. The pathogenic bacteria in the samples were cultured, isolated and identified, and the composition and drug susceptibility of pathogenic bacteria were analyzed. Results: 127 specimens were collected from 104 patients, among which sputum samples accounted for 45.67%, urine samples accounted for 31.50%, blood samples accounted for 15.75% and other samples accounted for 7.09%. A total of 127 duplicate bacterial strains were isolated from the samples, among which the gram-negative bacteria were the most, and Escherichia coli, Klebsiella pneumoniae and pseudomonas aeruginosa were the main ones. Staphylococcus aureus and Enterococcus faecium were the main gram-positive bacteria. The main fungi were candida albicans and Aspergillus. The resistance rates of Escherichia coli to ciprofloxacin, cefazolin and ampicillin were the highest, which were 94.55%, 89.09% and 74.55%, respectively. The resistance rates of Klebsiella pneumoniae to ciprofloxacin, cefazolin and acamicin were 86.96%, 82.61% and 82.61%, respectively. The resistance rates of P. aeruginosa to cefazolin, ciprofloxacin and levofloxacin were the highest, which were 73.33%, 66.67% and 60.00%, respectively. The resistance rates of Staphylococcus aureus to penicillin, gentamicin and erythromycin were 87.50%, 87.50% and 75.00%, respectively. Conclusion: The main pathogens of nosocomial infection were gram-negative bacteria, Escherichia coli, Klebsiella pneumoniae and pseudomonas aeruginosa. The drug resistance of different pathogenic bacteria is different to some extent, and rational drug use should be based on the drug resistance of patients.