Background: Ultrasound-guided lung biopsy (USLB) is a minimally invasive, real-time, safe and effective way to diagnose peripheral lung lesions. USLB is now widely used in clinical practice. However, the accuracy of USLB lacks large sample studies, and specimens from USLB are seldom used for microbial cultures. In this study, the efficiency of diagnosing tumours and infectious diseases with USLB is investigated, and the safety of USLB is evaluated. Methods: From September 2015 to August 2018, a total of 507 cases of USLB were retrospectively reviewed, including the pathological results, microbial culture results and complications. The final diagnosis was confirmed by clinical diagnosis and follow-up. The technical success rate and accuracy of USLB were calculated. Results: Six patients were excluded because they were lost to follow-up. A total of 501 patients were finally included (335 males and 166 females). The diagnostic accuracy of USLB was 82.0%. The accuracy of USLB in diagnosing lung tumours was 88.5% and that for diagnosing special infections was 55.2%. The positive rate of USLB lung tissue cultures was 18.3%. USLB can diagnose some microbial infections with high accuracy, such as mycobacterial infections, and Coptococcus neofotmans, Aspergillus fumigatus and Burkbolderia cepacia infections. Conclusions: USLB is a safe and effective method for diagnosing peripheral lung lesions with high accuracy and a low complication rate. Lung tissue pathology and cultures have good diagnostic value for tuberculosis and fungi infections. Lung tissue cultures can also diagnose bacterial infections and can contribute to the selection of antibiotics.