With an upsurge in demand for nuclear energy, chances of contamination of the environment, especially water have been raised by mine tailing and nuclear waste. For their advantages, uranium (U) mitigation with microbes, especially with biofilm, is of great interest, on top of chemical techniques. Biofilm is a microbial aggregation produced by most microbes and is an emerging tool for bioremediation for its multiple beneficial features. In this study, a bacterium was isolated from flooded water of rice field and found to be affiliated with well known prevalent freshwater biofilm forming genus Methylobacterium sp., and characterized for U removal potential from water using its biofilm. Significant loss of planktonic cell viability in presence of 100 mg L−1 U indicated its sensitivity towards U toxicity, however, it tolerated potentially toxic elements (PTEs) such as 10 mM Zn and Cd, 2 mM Cu and Co. The Gompertz model analysis, however, indicated greater impact of 50 µg L−1 over 100 µg L−1 U on maximum growth rate in TGY medium. The bacterium formed maximum biofilm in presence of 50 µg L−1 U, which ∼ 8-fold increased following use of 20 mM Ca2+. The grown biofilm showed higher phosphatase activity at pH 4.0 compared to pH 7.0 and 9.0. Following U amendment, increased U removal (90–96%) from type-I water in the pH range 4.0 to 7.0 was noticed compared to that from groundwater (60%) (pH 6.5) within 2 h at 30 °C. This decreased removal might be due to the presence of other co-ions in groundwater. U removal decreased by 3.4 to 12.9% in presence PTEs while Cu2+ and Co2+ impart more detrimental effect. Fluorescence microscopy and FE-SEM study indicated decreased cell viability and distorted cell morphology with external deposition of U, respectively, in the biofilm following U removal. The experimental data of biofilm-mediated U removal was found to follow the pseudo-second-order reaction kinetics and the Sips isotherm model. Thus, the present study provides a valuable insight into the development of cost-effective U removal methods from aquatic system using U-sensitive biofilm former.