Abstract This study aimed to evaluate the in vitro antifungal activity of terpinen-4-ol, tyrosol, and β-lapachone against strains of Coccidioides posadasii in filamentous phase (n = 22) and Histoplasma capsulatum in both filamentous (n = 40) and yeast phases (n = 13), using the broth dilution methods as described by the Clinical and Laboratory Standards Institute, to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of these compounds. The mechanisms of action of these compounds were also investigated by analyzing their effect on cell membrane permeability and ergosterol synthesis. The MIC and MFCf these compounds against C. posadasii, mycelial H. capsulatum, and yeast-like H. capsulatum, were in the following ranges: 350-5720 µg/mL, 20-2860 µg/mL, and 40-1420 µg/mL, respectively for terpinen-4-ol; 250-4000 µg/mL, 30-2000 µg/mL, and 10-1000 µg/mL, respectively, for tyrosol; and 0.48-7.8 µg/mL, 0.25-16 µg/mL, and 0.125-4 µg/mL, respectively for β-lapachone. These compounds showed a decrease in MIC when the samples were subjected to osmotic stress, suggesting that the compounds acted on the fungal membrane. All the compounds were able to reduce the ergosterol content of the fungal strains. Finally, tyrosol was able to cause a leakage of intracellular molecules.