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Background: Several case reports abound in literature about cases of histoplasmosis misdiagnosed as tuberculosis (TB). Nigeria is one of the highest TB-burdened countries, but data on histoplasmosis in Nigeria are sparse in the literature. The aim of this research was to investigate patients with presumptive pulmonary TB in Calabar, Nigeria, for histoplasmosis., Methods: This was a descriptive cross-sectional study of 213 participants with presumptive diagnosis of pulmonary TB between April 2020 and March 2021. Urine samples were collected from selected patients for Histoplasma antigen test using enzyme immunoassay kits, while sputum samples were collected for GeneXpert test for confirmed diagnosis of TB and conventional polymerase chain reaction (PCR) for the diagnosis of histoplasmosis., Results: Of the 213 participants enrolled into the study, 94 subjects (44.1%) were confirmed TB patients, 75 (35.2%) were human immunodeficiency virus (HIV) positive, 41 (19.2%) had advanced HIV disease (AHD), and 138 (64.8%) were HIV negative. Twenty-seven of the 213 participants were Histoplasma positive by antigen test and/or PCR, giving an overall prevalence rate of 12.7%. The prevalence of histoplasmosis among confirmed TB patients (7.4% [7/94]) was significantly lower than in unconfirmed TB patients (16.8% [20/119]) ( P  = .04). Participants on anti-TB therapy also had a significantly lower rate of histoplasmosis compared to those not on anti-TB drugs ( P  = .00006). The internal transcribed spacer (ITS) sequencing of the Histoplasma revealed a closely relatedness to Histoplasma capsulatum., Conclusions: Histoplasmosis is not uncommon among presumptive TB patients. There should be proper microbiological investigation of patients presenting with symptoms suggestive of TB to exclude cases of histoplasmosis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)