Various models have been proposed to explain the formation mechanism of colloform sphalerite, but the origin is still under debate. In order to decipher influences on trace element incorporation and sulfur isotope composition, crystalline and colloform sphalerite from the carbonate-hosted Mississippi-Valley Type (MVT) deposit near Wiesloch, SW Germany, were investigated and compared to sphalerite samples from 52 hydrothermal vein-type deposits in the Schwarzwald ore district, SW Germany to study the influence of different host rocks, formation mechanisms and fluid origin on trace element incorporation. Trace and minor element incorporation in sphalerite shows some correlation to their host rock and/or origin of fluid, gangue, paragenetic minerals and precipitation mechanisms (e.g., diagenetic processes, fluid cooling or fluid mixing). Furthermore, crystalline sphalerite is generally enriched in elements like Cd, Cu, Sb and Ag compared to colloform sphalerite that mainly incorporates elements like As, Pb and Tl. In addition, sulfur isotopes are characterized by positive values for crystalline and strongly negative values for colloform sphalerite. The combination of trace element contents, typical minerals associated with colloform sphalerite from Wiesloch, sulfur isotopes and thermodynamic considerations helped to evaluate the involvement of sulfate-reducing bacteria in water-filled karst cavities. Sulfate-reducing bacteria cause a sulfide-rich environment that leads in case of a metal-rich fluid supply to a sudden oversaturation of the fluid with respect to galena, sphalerite and pyrite. This, however, exactly coincides with the observed crystallization sequence of samples involving colloform sphalerite from the Wiesloch MVT deposit.