To investigate the positive rate, species and drug resistance of Mycobacteria in clinical specimens of acquired immunodeficiency syndrome (AIDS) patients, the AIDS patients in a hospital in Guangxi during January 2010 and December 2019 were collected and their clinical specimens were cultured with acid-fast bacilli. The antimicrobial susceptibility testing was conducted with more than eight anti-tuberculosis drugs after isolation and identification. The overall positive rate was 15.68% (2 163/13 795). The positive rate was high in pus, secretions, various tissue specimens, pleural effusion, and ascites. The species identification results showed that Mycobacteria tuberculosis compound group accounted for 77.95% (1 442/1 850) and non-tuberculosis Mycobacterium (NTM) accounted for 22.05% (408/1 850). The latter accounted for 15.71%-26.07% over 10 years, with no statistically significant difference between years (χ2＝10.442, P>0.05). The overall drug resistance rate was 23.30% (336/1 442), with a significant difference over a 10-year period (χ2＝18.901, P＝0.026). The rates of resistance to isoniazid, rifampicin, streptomycin, ofloxacin, ethambutol, p-amino salicylic acid, kanamycin were 12.14% (175/1 442), 10.54% (152/336), 9.29% (134/1 442), 5.62% (81/1 442), 3.05% (44/1 442), 1.80% (26/1 442), 1.32% (19/1 442), respectively. The prevalence of multidrug-resistant tuberculosis (DR-TB) and extensively drug-resistant tuberculosis (XDR-TB) was 5.48% (79/1 442) and 0.28% (4/1 442), respectively. It is confirmed that the positive rate of Mycobacteria isolated from the clinical specimens of AIDS patients is high and NTM accounts for a high proportion. The drug resistance rate of Mycobacterium tuberculosis to commonly used anti-tuberculosis drugs is high. It is suggested that clinical Mycobacterium identification and antimicrobial susceptibility testing should be carried out to provide a reliable basis for clinical diagnosis and treatment.