The surface properties of fluorite are often affected by dissolved gangue species (e.g., calcite) during the flotation process. Microflotation testing with and without the addition of calcite supernatant was conducted using octanohydroxamic acid (OHA) as the collector. The results revealed that dissolved calcite species significantly affected the flotation behavior of fluorite. Fourier transform infrared spectra confirmed that the decrease in flotation recovery was linked to lower OHA adsorption. Solution chemistry analysis indicated that CaCO₃ and Ca²⁺ from the calcite supernatant were the most favorably adsorbed species, and X-ray photoelectron spectroscopy analysis confirmed the surface adsorption of calcite species. Density functional theory simulations provided a detailed analysis of the multidentate adsorption configuration of OHA, which was the most favorable for adsorption on the fluorite surface. The adsorption energy calculation showed that the calcite dissolved species were more stably adsorbed on the fluorite surface than OHA. The pre-adsorption of calcite dissolved species hindered the adsorption of OHA due to electrostatic repulsion.