A large number of recalcitrant bacterial pathogens cannot be easily treated by antibiotics due to the existence of biofilm. Hence, an alternative strategy needs to be adopted to remove the biofilm without the development of antibiotic resistance. Bacteriocins, ribosome-mediated proteinaceous toxins, having potential to inhibit the growth of closely or distantly related bacteria. In the present study, after screening a number of sources, a bacteriocin-producing strain, Enterococcus faecalis BDR22, was isolated that showed a significant reduction in the growth of planktonic cells of Gram-positive Staphylococcus aureus, Bacillus subtilis, and Gram-negative Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Enterobacter cloacae, and Klebsiella pneumoniae compared to the conventional antibiotic tetracycline. The considerable reduction of the biofilm-forming sessile cells of the test organisms S. aureus (ATCC 23235) and P. aeruginosa (ATCC 10145), with no significant cell revival even after withdrawal of the treatment, was also observed. The extracellular polymeric substance (EPS) content of the biofilm was also reduced, with around 84% total carbohydrate reduction found for both microorganisms. The antibiofilm activities of the strain against test organisms were clearly visible from scanning electron micrographs and confirmed by the changes in functional groups (C-H, -OH, C = C, C-N etc.) of biofilm matrices by Fourier transform infrared spectroscopy (FTIR) analysis. The molecular docking interactions with docking energies ∆G of - 54.40 kcal/mol and - 66.2373 kcal/mol validate the affinity of the bacteriocin towards the biofilm-forming protein, which confirms the competence of the bacteriocin-producing strain to act as an effective antimicrobial and antibiofilm agent, replacing antibiotics.