Targeted next-generation sequencing (tNGS) might be valuable for identifying disease-causing pathogens. Herein, we assessed the utility of tNGS in diagnosing pulmonary infections using sputum samples. We gathered complete clinical information and tested the specimens using both conventional microbiological tests (CMTs) and tNGS. The goal was to compare the effectiveness of these two methods in detecting viral, bacterial, and fungal pathogens. Notably, tNGS demonstrated a higher pathogen detection rate compared to CMTs (80.26 % [122/152] vs. 33.55 % [51/152], P = 0.029). Specifically, tNGS was more effective in detecting viruses than CMTs (90.00 % vs. 28.07 %, P = 0.003). Moreover, tNGS detected certain fungi, such as Candida albicans and Cryptococcus neoformans, although the difference between the two assays was not statistically significant (P >
0.05). Our findings reveal that tNGS offers significant advantages in detecting pathogens in patients with lung infections, particularly for bacteria and viruses, providing valuable information that complements CMTs.