Brain mass lesions (BML) occurred in 10% of patients infected by the human immunodeficiency virus (HIV), as expression of severe and sometimes treatable diseases. However, the management of them is not well established. We analyzed, retrospectively, 26 brain biopsies (22 estereotaxic) in patients with HIV infection and BML to know their usefulness and safety. The inclusion criteria were: Failure of the anti-Toxoplasma empirical treatment, atypical scan appearance for toxoplasmosis, or severe neurological picture. Brain biopsy yielded a diagnosis in 19 patients (73.1%): Progressive multifocal leukoencephalopathy (n = 8; 30.8%), primary central nervous system lymphoma (n = 6; 23.1%), mycobacteriosis (n = 2; 7.7%), toxoplasmosis (n = 2; 7.7%), criptococcosis (n = 1), cryptosporidiosis (n = 1) and HIV-encephalitis (n = 1). In one case there was a multiple diagnosis: mycobacteriosis, toxoplasmosis, and lymphoma. Brain biopsy results decided a change in therapy in 65.4%, the resolution or improvement of the neurological process in 30.8%, and the determination of the prognosis in 30.8%. In 8 cases (30.7%) there were biopsy complications, with secondary mortality in one. Brain biopsy of BML in HIV-infected patients is a diagnostic method with a high overall diagnostic rentability and uncommon non reversible complications, offering the possibility to prescribe a specific and potentially curative treatment.