BACKGROUND: Urine antigen testing is a rapid sensitive method for detecting active infection with the endemic fungi Histoplasma capsulatum and Blastomyces dermatitidis. Herein, we compared the performance of the MiraVista Diagnostics (MVista) Histoplasma urine antigen assay with the Niche Diagnostics (ND) Histoplasma urine antigen assay for the detection of histoplasmosis and blastomycosis. METHODS: Two hundred fifty urine samples from 234 patients previously tested by the MVista Histoplasma urine antigen assay as part of routine care were tested by the ND Histoplasma and Blastomyces urine antigen assays. The electronic medical records of all patients whose samples were tested were retrospectively reviewed to identify patients with a clinical diagnosis of and/or treatment for histoplasmosis or blastomycosis, and the diagnostic workup undertaken to support these diagnoses. RESULTS: The MVista and ND Histoplasma urine antigen assays were highly concordant, showing 99% overall agreement (90.5% positive agreement and 99.6% negative agreement). Three specimens collected after antifungal therapy returned discrepant results, with the MVista assay positive in 2 of these and the ND assay positive in 1; in each case, the antigen concentration was near the lower quantification limit. Both Histoplasma assays were positive in all patients with culture-proven blastomycosis (n = 3). CONCLUSIONS: The MVista and ND Histoplasma urine antigen assays performed similarly in identifying histoplasmosis cases encountered in routine clinical practice, with discrepancies affecting posttreatment specimens. Given the paucity of Blastomyces-positive samples, further studies are needed to better compare the utility of the MVista and ND Histoplasma urine antigen assays in diagnosing blastomycosis.