In response to increasing anthropogenic pollution and metal accumulation in aquatic environments, bioremediation has gained great importance. Microbial consortia are emerging as a promising solution due to their diverse pollutant metabolizing pathways. This study evaluates the role of biotic and abiotic components in microbial mats for Cr(VI) removal from freshwater. For this purpose, microbial mats were modified by high-temperature treatments using an autoclave and a muffle furnace, and modified and unmodified mats, both chemically characterized by SEM-EDS, ATR-FTIR, XRD, and XPS, were used in Cr(VI) removal assays. The concentrations of total Cr and Cr(VI) were measured, which were initially 15 mg/L and after 17 days reached values between 0.22 and 4.99 mg/L depending on the treatment. The removal efficiency was nearly 99% in unmodified mats, while in autoclaved ones it reached 95% and in calcined mats 67%. These findings demonstrate that all components contribute to Cr removal. Both trivalent and hexavalent Cr penetrated the porous structure reaching deep layers where they were adsorbed by electrostatic attraction or complex formation with the mat components, while the insoluble compounds formed, such as Cr