Objective To explore the effect of comprehensive intervention measures on improving the pathogen detection rate before antimicrobial treatment as well as their impact on the structure of antimicrobial use and pathogen resistance in secondary hospitals. Methods A before-and-after controlled study was adopted. Information on pathogen detection, antimicrobial use, and pathogen resistance surveillance of clinical departments of a secondary first-class general hospital from January to December 2023 (before intervention) and from January to December 2024 (after intervention) were collected, including pathogen detection data, antimicrobial use data, and pathogen resistance surveillance data from each department. Relevant surveillance indicators before and after the implementation of comprehensive intervention measures were compared. Results After intervention, pathogen detection rate of the whole hospital increased from 27.45% to 57.48% (P<0.001), with particularly significant improvement in the intensive care unit (ICU) and department of pediatrics, increasing from 33.44% and 23.15% to 75.04% and 85.10%, respectively. Antimicrobial use presented a structural adjustment, with consumption of cefotaxime decreasing by 48.2% (from 15.51 DDD/100 person-day to 8.03 DDD/100 person-day) and consumption of piperacillin/tazobactam increasing by 69.0% (from 6.39 DDD/100 person-day to 10.80 DDD/100 person-day). Resistance rates of major pathogens decreased. The resistance rates of Escherichia coli to amoxicillin/clavulanic acid and ceftazidime decreased from 31.44% and 45.23% to 20.73% and 32.40%, respectively (both P<0.05). The resistance rate of Klebsiella pneumoniae to meropenem decreased from 3.91% to 1.81% (P<0.05). Conclusion Comprehensive intervention measures can effectively improve the pathogen detection rate in secondary hospitals, optimize the structure of antimicrobial use, reduce the resistance rate of major pathogens, and have important practical significance for achieving refined management on antimicrobial agents in secondary hospitals.