Objective To investigate the distribution and drug resistance of pathogens in bronchoalveolar lavage fluid (BALF) samples in our hospital, so as to provide reference for clinical rational drug use. Methods Pathogens were isolated and identified from 615 BALF samples between Jan. 2016 and Dec. 2019 in our hospital, and drug sensitivity test in vitro was conducted. Results A total of 707 non repetitive pathogens were detected, including 593 (83.9%) Gram-negative bacilli and 114 (16.1%) Gram-positive bacilli. The top 3 pathogens were Pseudomonas aeruginosa (171 strains, 24.2%), Klebsiella pneumoniae (147 strains, 20.8%) and Acinetobacter baumannii (97 strains, 13.7%), respectively. Among the Gram-negative bacilli, Pseudomonas aeruginosa had a higher resistance rate to carbapenems, while the resistance rates to imipenem and meropenem were 33.3% (57/171) and 30.6% (52/170), respectively; Klebsiella pneumoniae had a lower resistance rate to carbapenems (<20%), the extended-spectrum β-lactamase (ESBL) production strains in Klebsiella pneumoniae was 16.3% (24/147); and Acinetobacter baumannii had a higher resistance rate of 85.6% (83/97) to imipenem and meropenem. Among the Gram-positive bacilli, the resistance rate of Staphylococcus aureus to penicillin was 89.4% (76/85). Gram-positive pathogens were not yet resistant to vancomycin and linezolid. The detection rate of methicillin-resistant Staphylococcus aureus was 32.9% (28/85). Conclusion The main pathogens isolated from BALF samples in our hospital are Gram-negative bacilli, and their drug resistance can not be ignored. The detection of pathogens in BALF has high clinical value for disease diagnosis and rational use of antibiotics.