Abstract: OBJECTIVE To investigate the clinical application value of targeted high-throughput sequencing (tNGS) technology in the diagnosis of mixed infections with multiple pathogens in alveolar lavage fluid from patients with pulmonary tuberculosis, and to analyze the pathogen spectrum characteristics and clinical significance of mixed infections. METHODS A retrospective analysis was conducted on the tNGS results of 198 respiratory pathogens in alveolar lavage fluid samples from 146 patients confirmed with pulmonary tuberculosis, who were admitted to the Tuberculosis Infection Ward of Red Cross Hospital of Yulin City from Jan. 1, 2024 to Dec. 31, 2024. These results were compared with the pathogen detection rates and the composition of mixed infections obtained through traditional detection methods (including smear, culture and rapid etiological detection). RESULTS Among the alveolar lavage fluid samples from 146 patients, the positive rate of pathogens detected by tNGS was 96.57%, while that detected by traditional methods was 54.11%, showing a statistically significant difference (χ²=70.861, P<0.001). tNGS detected mixed infections with multiple pathogens in 91 cases (62.33%), with the main mixed infectious pathogens including bacteria such as Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, fungi such as Candida albicans, Candida tropicalis, Pneumocystis jirovecii and Aspergillus fumigatus, and viruses such as Epstein-Barr virus, cytomegalovirus and rhinovirus. The common mixed infection patterns and their proportions were bacteria+virus (28.57%), bacteria+fungi (36.26%) and mixed infections with all three (bacteria+fungi+virus) (12.09%). The mixed infection patterns tended to become more complex with increasing age across different age groups. CONCLUSION tNGS technology can effectively identify mixed infections with multiple pathogens in alveolar lavage fluid from patients with pulmonary tuberculosis, providing comprehensive pathogen spectrum information and serving as an important basis for precise clinical anti-infective treatment.