A microbial treatment of fluorinated aqueous solution was performed by using a novel bacterial strain isolated from fluoride‐loaded groundwater. Defluoridation efficiencies of two forms viz. immobilized live cells and dead biomass of the isolate were investigated by means of a series of batch studies. Initial fluoride concentration (1–25 mg L−1), pH (5–9), bead numbers (5–30), agitation speed (100–240 rpm), and temperature (20–45°C) were the various parameters studied to investigate the influence of each of them on fluoride removal capacity of the microbial species. The bacterial species isolated was able to withstand fluoride concentration till 20 mg L−1 in case of immobilized live cells, whereas, the dead biomass could reduce the concentration only up to 10 mg L−1. 16S rRNA sequencing confirmed the isolate to be Staphylococcus lentus. Biochemical characterizations were performed. Biokinetics of fluoride uptake by the isolate was performed. Instrumental analysis such as scanning electron microscopy and FTIR substantiated the uptake of the fluoride ions by the biomass. Maximum removal of 92% and 85.03% were achieved by the immobilized cells and dead biomass respectively which is the highest reported till date. Maximum sorption capacity of 5.198 mg g−1 of the dead cells were obtained from the Langmuir isotherm. Adsorption equilibria was found to be best explained by the Freundlich isotherm model. Pseudo‐second‐order kinetics provided the best fit to the fluoride‐sorption system. Experimental results revealed that the immobilized cells of Staphylococcus lentus (KX941098) could be used as potential agents in removal of fluoride from contaminated groundwater. © 2018 American Institute of Chemical Engineers Environ Prog, 37: 1573–1586, 2018