In this study, Staphylococcus aureus biofilms, which are considered a foe for being pathogenic, were tested for their uranium bioremediation capacity to find out if they can turn out to be a friend. Acid phosphatase activity, which is speculated to aid in bio-precipitation of U(VI) from uranyl nitrate solution, was assayed in biofilms of seven different S. aureus strains. The presence of acid phosphatase enzyme was detected in the biofilms of all S. aureus strains (in the range of 3.1 ± 0.21 to 26.90 ± 2.32 μi.u./g), and found to be higher when compared to that of their planktonic phenotypes. Among all, S. aureus V329 biofilm showed highest biofilm formation ability along with maximum phosphatase activity (26.9 ± 2.32 μi.u./g of biomass). Addition of phosphate enhanced the U(VI) remediation when treated with uranyl nitrate solution. S. aureus V329 biofilm showed significant U tolerance with only a 3-log reduction when exposed to 10 ppm U(VI) for 1 h. When treated in batch mode, V329 biofilm successfully remediated up to 47% of the 10 ppm U(VI). This new approach using the acid phosphatase from the S. aureus V329 biofilm presents an alternative method for the remediation of uranium contamination.