Mercury pollution is a significant environmental issue, primarily resulting from industrial activities, including gold mining extraction. In this study, 333 microorganisms were tested in increasing mercury concentrations, where 158 bacteria and 14 fungi were able to grow and remain viable at concentrations over 5.0 mg/L of mercuric chloride (II). One of the bacterial strains, Stenotrophomonas sp. INV PRT0231, isolated from the mouth of the San Juan River in the Chocó region in Colombia, showed a high mercury resistance level (MIC₉₀ of 27 ± 9 mg/L), with a removal rate of 86.9%, an absorption rate of 1.2%, and a volatilization rate of 85.7% at pH 6.0 and 30.0 °C. The FTIR analysis showed changes in the functional groups, including fatty acid chains and methyl groups, proteins, and lipopolysaccharides associated with the carboxylate group (COO⁻), suggesting an important role of these biomolecules and their associated functional groups as mechanisms employed by the bacterium for mercury detoxification. Our study contributes to the understanding of the mechanisms of mercury biotransformation in microbial environmental isolates to help develop bioremediation strategies to mitigate mercury pollution caused by anthropogenic activities.