Background Gene chip technology has been increasingly used in the diagnosis and treatment of common tuberculosis. However, its role in the diagnosis and treatment of silicosis complicated with mycobacterial infection remains unclear. Objective To evaluate the application value of gene chip technology in the diagnosis and treatment of silicosis complicated with mycobacterial infection. Methods From January 2019 to June 2021, 197 silicosis patients suspected to be complicated with mycobacterial infection in Quanzhou First Hospital Affiliated to Fujian Medical University were enrolled in this study. The etiology evaluation for the 197 patients was conducted by acid-fast staining of sputum smear (sputum smear method), culture of Mycobacterium tuberculosis of sputum (sputum culture method), and gene chip technology of bronchoalveolar lavage fluid (BALF); and for 80 patients among them, acid-fast staining of BALF (BALF smear method) and culture of Mycobacterium tuberculosis of BALF (BALF culture method) were additionally performed. The positive rates and consistency were assessed using intraclass correlation coefficient (ICC). Test for Mycobacterium tuberculosis drug resistance mutation gene was added for patients with Mycobacterium tuberculosis complex by BALF gene chip technology. Results The average age of the 197 patients was (53.1±9.1) years, and the average dust exposure time was (21.1±9.4) years, including 192 males and 5 females. There were 8 cases with stage I silicosis, 17 cases with stage II silicosis, and 172 cases with stage III silicosis. Among them, 11.2% were positive for sputum smear; 24.4% were positive for sputum culture, and 36.0% were positive by gene chip of BALF. The difference between the three methods was statistically significant (P<0.05). The result of consistency test for the three methods showed that the ICC was 0.539 (P<0.001). Among the 80 patients, there was a significant difference in the positive rates of the five methods (χ²=25.23, P<0.001). The results of Bonferroni test showed statistically significant pair-wise differences between BALF culture method and sputum smear method, BALF culture method and BALF smear method, BALF gene chip method and sputum smear method, BALF gene chip method and BALF smear method (P<0.05), while there were no statistically significant differences between the other pairs (P>0.05). The result of consistency test for the five methods showed that the ICC was 0.586 (P<0.001). Among the 71 BALF gene chip positive cases, 59 cases reported positive Mycobacterium tuberculosis complex (17 cases were positive in the first-line anti-tuberculosis resistance test, and 2 cases were found positive quinolone resistance gene in the second-line anti-tuberculosis resistance test), and received regular anti-tuberculosis treatment, among them 45 cases improved and 14 cases were stable; 12 cases reported non-tuberculous mycobacteria cases, among them 5 cases received anti-non-tuberculous mycobacteria treatment (4 cases improved and 1 case was stable), and 7 cases with mild symptoms did not receive anti-non-tuberculous mycobacteria treatment. Conclusion Compared with sputum smear, sputum culture, and other traditional methods, gene chip technology of BALF can improve the positive rate of pathogenic diagnosis of silicosis complicated with mycobacterial infection, and can also quickly identify whether it is non-tuberculous mycobacteria infection or drug-resistant Mycobacterium tuberculosis infection, which is helpful to adjust treatment as soon as possible.