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Background: Opportunistic infections in the central nervous system (CNS) of people with HIV/AIDS (PLWHA) remain significant contributors to morbidity and mortality, especially in resource-limited scenarios. Diagnosing these infections can be challenging, as brain imaging is non-specific and expensive. Therefore, molecular analysis of cerebrospinal fluid (CSF) may offer a more accurate and affordable method for diagnosing pathogens., Methods: We conducted extensive real-time PCR testing (qPCR) on CSF to evaluate etiological agents in PLWHA with neurological manifestations. Primers targeting DNA from specific pathogens, including cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), John Cunningham virus (JCV), Toxoplasma gondii , and human T-lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2), were used., Results: Cerebrospinal fluid samples revealed 90 pathogens (36.7%). Toxoplasma gondii was the most frequently detected pathogen, found in 22 samples (30.5%). Other pathogens included Cryptococcus sp. (7.7%), EBV (5.3%), CMV, VZV, and JCV (4.0% each)., Conclusion: Despite antiretroviral therapy and medical follow-up, opportunistic central nervous system infections remain frequent in PLWHA. Herpesviruses are commonly detected, but T. gondii is the most prevalent opportunistic pathogen in our study population. Therefore, molecular diagnosis is a crucial tool for identifying opportunistic infections, even in patients undergoing treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 de Melo, Pinto, Ferreira, Brotas, Marinho, da Silva, Monte, Feitoza, Reis, Almeida, Ferreira and Bastos.)