Background: This study aimed at improving a real-time polymerase-chain-reaction (qPCR) assay for the detection of Histoplasma capsulatum, a fungal pathogen that can cause severe respiratory infections in humans, in clinical and soil samples. Methods: Primer and probes were in-silico designed, in-silico and in-vitro evaluated including clinical biopsy materials and finally subjected to a realworld application with collected soil samples. Results: Applying the qPCR assay with liver and lung biopsies from 71 patients each, including 59 patients infected with human immunodeficiency virus (HIV), as well as with Sabouraud (SAB) agar culture as the diagnostic reference standard, diagnostic accuracy of the qPCR assay of 100% (5/5) sensitivity and 96% (63/66) specificity for liver samples and 100% (4/4) sensitivity and 94% (63/67) specificity for the lung samples was recorded. When applying the assay with soil samples from caves near of Presidente Figueiredo city, Amazonas, Brazil, one sample from the Maroaga cave was confirmed as positive. Conclusions: The improved qPCR assessed in this study was successful in detecting H. capsulatum with high efficiency and accuracy in in-vitro evaluation, including the identification of the target pathogen in both clinical and environmental samples.  Kulcsszavak: in-silico evaluation, in-vitro evaluation, Histoplasma capsulatum, qPCR, sensitivity, specificity, biopsy, soil, Presidente Figueiredo caves