AbstractBackgroundCulture can be used for diagnosis and antifungal susceptibility testing in animals with fungal infections. Limited information is available regarding the diagnostic performance of culture and the susceptibility patterns ofHistoplasmaspp. isolates.Hypothesis/ObjectivesDescribe the clinical utility of culture and the susceptibility patterns ofHistoplasmaspp. isolates causing histoplasmosis in cats and dogs.AnimalsSeventy‐one client‐owned animals, including 33 cats and 19 dogs with proven or probable histoplasmosis.MethodsCulture was attempted from tissue or fluid samples. Diagnostic performance of culture, cytopathology, and antigen detection were compared with final diagnosis. Susceptibility to antifungal agents was determined for a subset (11 from dogs, 9 from cats) of culture isolates.ResultsCulture had a diagnostic sensitivity of 17/33 (52%; 95% confidence interval [CI], 34%‐69%) and 15/19 (79%; 95% CI, 61%‐97%) and specificity of 6/6 (100%; 95% CI, 54%‐100%) and 10/10 (100%; 95% CI, 69%‐100%) in cats and dogs, respectively. Culture was not positive in any animal in which cytopathology and antigen testing were negative. Target drug exposure (area under the concentration curve [AUC]/minimum inhibitory concentration [MIC] >25) should be easily achieved for all isolates for itraconazole, voriconazole, or posaconazole. Five of 20 (25%) isolates had fluconazole MIC ≥32 μg/mL and achieving target drug exposure is unlikely.Conclusions and Clinical ImportanceFungal culture did not improve diagnostic sensitivity when used with cytopathology and antigen detection. Susceptibility testing might help identify isolates for which fluconazole is less likely to be effective.