Histoplasmosis is a life-threatening opportunistic infection in individuals with advanced HIV disease (AHD), particularly in endemic regions such as Latin America. Early diagnosis is crucial for reducing mortality but remains underdiagnosed due to non-specific clinical presentations and limited diagnostic access. This study evaluates the impact of systematic histoplasmosis screening over 18 months (March 2021-September 2022) using the Histoplasma urinary antigen detection test in people living with HIV (PLHIV) receiving outpatient care or hospitalized at a reference center in Southern Brazil. A retrospective analysis was conducted on PLHIV screened with the Clarus® Histoplasma GM enzyme immunoassay (HGM-EIA, IMMY, USA). Inclusion criteria comprised a recent HIV diagnosis (≤30 days), poor adherence to or abandonment of antiretroviral therapy (>
90 days), CD4 + count <
200 cells/mm³, or clinical/radiological findings suggestive of histoplasmosis. Clinical, laboratory, and treatment data were assessed, along with 90-day outcomes. Among 287 PLHIV screened, Histoplasma antigen was detected in 9.1% (26/287), including 19.6% of hospitalized patients and 6.4% of outpatients. Overall mortality was 24%, with a higher rate among inpatients (27.3%). Fever, neurological impairment, and lung opacification were more frequent in hospitalized cases. Amphotericin B deoxycholate was more commonly administered to inpatients (63.6% vs. 25%). Mortality at 90 days was significantly higher in patients with a histoplasmosis case-fatality score ≥ 5 (66.7% vs. 33.3%, p = 0.016). Systematic screening enhances early detection of histoplasmosis, facilitating timely treatment and reducing hospitalizations and mortality.