Background: Reports on the application of metagenomic next-generation sequencing (mNGS) to diagnose infectious peripheral pulmonary lesions (PPLs) remain scarce. No research has ever explored which specimen is preferred for mNGS. Methods: We applied mNGS to detect the presence of pathogen in matched transbronchial lung biopsy (TBLB), bronchoalveolar lavage fluid (BALF) and bronchial needle brushing (BB) specimens from 39 patients suspected of having infectious PPLs. We explored the differences in the composition and diagnostic efficacy of the three specimens. Results: We found that mNGS of TBLB, BALF, BB and TBLB + BALF + BB was more sensitive than conventional cultures for bacterial infections, fungal infections and general infections, the difference in sensitivity of mNGS between TBLB, BALF, and BB was not statistically significant. The sensitivity of mNGS of TBLB + BALF + BB for the diagnosis of fungal infections or general infections was higher than mNGS of TBLB and not significantly higher than mNGS of BALF or BB. We found that there was no statistically significant difference in the relative abundance of pathogen amongst the three types of specimens and in relative abundance of all of the six kinds of common oropharyngeal microbiota between TBLB and BB specimens. Conclutions: The study indicated that mNGS of TBLB, BALF or BB could yield a higher sensitivity for pathogen identification. mNGS of the BB samples might be an alternative choice for patients with infectious PPLs but couldn’t tolerate more invasive TBLB procedures.