Groundwater fluoride contamination is one of the most serious toxicological environmental issues in India and around the world. Water samples were taken from Natham taluk and screened for fluoride resistant bacteria. Initially, twenty-four fluoride resistant colonies were selected from 50 mM NaF supplemented LB agar plates. On blood agar plates, all isolates showed β-haemolysis, confirming their status as pathogens. Virulence factors (algD, plcH, toxA, gyrB, rhlC, lasB) and biofilm-forming genes (ppyR, pelA, pslA) were identified in these isolates by PCR analysis. The fluoride ion transporter ‘crcB’ was successfully amplified from these isolates by gene-specific PCR. Genus Pseudomonas and P. aeruginosa species-specific PCR analysis identified that all strains as belonging to the P. aeruginosa species. Besides, three high fluoride resistance strains were selected based on high fluoride resistance and confirmed as P. aeruginosa species by 16S rRNA sequencing and NCBI blast analysis.