Objective To explore the effect of applying comprehensive interventions on promoting pathogen detection before antimicrobial therapy in hospitalized patients. Methods Hospitalized patients who received therapeutic use of antimicrobial agents in a tertiary first-class hospital from January 2020 to December 2021 were selected as the research subjects. Comprehensive intervention measures were implemented from January 2021. The pathogen detection rates, detection classification, and detection rates of key monitored departments before antimicrobial therapy were compared between the pre-intervention group (January-December 2020) and the post-intervention group (January-December 2021). Results A total of 10 239 hospitalized patients who received therapeutic use of antimicrobial agents were included in analysis, 4 526 cases were in the pre-intervention group and 5 713 cases in the post-intervention group. The pathogen detection rates before antimicrobial therapy, before restricted grade antimicrobial therapy, and before special grade antimicrobial therapy after intervention were 94.56%, 94.72%, and 96.03%, respectively, which were higher than 83.74%, 84.47%, and 84.95% before intervention, with statistical significance (all P < 0.05). The detection rate of targeted pathogens after intervention was 64.87%, higher than that before intervention (28.04%), with statistically significant difference (P < 0.05). The pathogen detection rates before therapeutic use of antimicrobial agents in departments of critical care medicine, pulmonary and critical care medicine, pediatrics, neurosurgery, and general surgery after intervention were 93.20%, 91.17%, 92.20%, 94.12%, and 91.15%, respectively, higher than the rates before intervention, namely 85.00%, 82.19%, 83.20%, 83.33%, and 83.03%, respectively, with statistical significance (all P < 0.05). Conclusion The application of comprehensive intervention measures can improve the pathogen detection rate before antimicrobial therapy of hospitalized patients. Close attention should be paid to the pathogen detection indicators related to healthcare-associated infection diagnosis and for the detection of sterile body fluid.