Objective To understand the pathogen detection of hospitalized patients before antimicrobial therapy in a hospital through implementation of comprehensive intervention measures, and provide reference basis for the development of targeted measures. Methods Hospitalized patients who received therapeutic antimicrobial agents in this hospital were selected as the research subjects. Patients who were hospitalized from January to May 2022 were selected as the pre-intervention group, comprehensive intervention measures were taken from June to October 2022, and those who were hospitalized from November 2022 to March 2023 were selected as the post-intervention group. The pathogen detection rate before antimicrobial therapy, sterile specimen detection rate, antimicrobial use rate, detection rate of key multidrug-resistant organisms of patients before and after the intervention were analyzed. Results Compared to before intervention, the proportion of pathogen detection rate before antimicrobial therapy (62.09% vs 74.04%), detection rate of healthcare-associated infection diagnosis-related pathogens (62.82% vs 92.73%), and sterile specimen detection rate (35.17% vs 41.06%) of hospitalized patients after intervention all increased significantly, with statistically significant differences (all P < 0.05). After intervention, pathogen detection rate before the combination use of key antimicrobial agents was not statistically different from before intervention (93.33% vs 90.48%, P>0.05), while antimicrobial use rate was lower than before intervention (39.93% vs 44.95%, P < 0.05). There was no statistically significant difference in the detection rate of key multidrug-resistant organisms before and after intervention (all P>0.05). Conclusion Adopting scientific and rational intervention measures can improve the pathogen detection rate, provide a reference basis for the rational use of antimicrobial agents. There was no significant improvement in the pathogen detection rate before the combination use of key antimicrobial agents and the detection rate of key multidrug-resistant organisms, indicating that relevant measures still need to be further optimized.