Pseudomonas aeruginosa (P. aeruginosa), a Gram-negative opportunistic pathogen, poses significant treatment challenges due to its antibiotic resistance and biofilm formation. This study investigates the anti-bacterial and anti-biofilm activities of chemically synthesized selenium nanoparticles (SeNPs) against P. aeruginosa. SeNPs were synthesized using ascorbic acid as a reducing agent and characterized. Biofilm formation was quantified using a modified microtiter plate method, and the anti-biofilm efficacy of SeNPs was evaluated using confocal microscopy and SEM. The P.aeruginosa isolates exhibited high resistance to piperacillin-tazobactam (60%) and ceftazidime (59%). SeNPs demonstrated a round shape with a diameter of 15-18 nm. UV-Vis spectra showed a peak at 275 nm, and XRD analysis revealed crystalline peaks corresponding to selenium. The FTIR spectra confirmed the presence of various functional groups. SeNPs significantly reduced biofilm formation in a dose-dependent manner, with MIC50 and MIC90 values of 60 μg/mL and 80 μg/mL, respectively. Confocal microscopy and SEM analysis showed a notable decrease in biofilm thickness and bacterial adherence post-SeNPs treatment. These findings suggest that SeNPs could be a promising alternative or adjunctive treatment option for combating antibiotic-resistant P. aeruginosa infections. Further research is warranted to explore the clinical applications of SeNPs in treating biofilm-associated infections.