BACKGROUND: The rapid and accurate identification of infectious pathogens in HIV-infected patients remains a challenge. Metagenomic next-generation sequencing (mNGS) is a panpathogen assay for rapid diagnosis of infectious diseases. Here, the diagnostic value of mNGS was evaluated in HIV-infected patients and compared with conventional microbiological tests (CMTs). METHODS: This study was conducted on 216 hIV-infected patients with suspected opportunistic infections. Infectious pathogen detection was done by mNGS and conventional microbiological tests, respectively. RESULTS: A total of 195 patients (90.2%) were positive for microbial pathogens by mNGS, while 135 patients (62.5%) were positive for microbial pathogens by CMTs. Mixed infection was identified in 92 patients by mNGS, and 41 patients were detected with mixed infection by CMTs. Fungi and virus mixed infection was the most frequent pattern detected by mNGS (32, 14.8%) and CMTs (22, 10.2%). The CD4+ T cell count in patients with mixed pathogens was significantly lower than that in patients infected with a single pathogen. Pathogens were quickly identified by mNGS in 151 patients (69.9%), and appropriate treatments were initiated. In 47 patients antibacterial agents were adjusted based on mNGS results, in 39 patients antifungal agents were changed, and 35 patients had antiviral agents added. CONCLUSION: mNGS is a valuable tool and enhances rapid microbiological identification in HIV-infected patients. Combined with CMTs, mNGS may facilitate personalized antimicrobial treatment strategies and increase survival.