<p>Biofilm formation presents significant challenges in healthcare, food processing, and water treatment, contributing to antibiotic resistance and persistent infections. Effective strategies to combat biofilm-associated infections are urgently needed. This study introduces a novel approach to biofilm removal by bio-functionalizing T4 bacteriophage with modified Fe₃O₄ nanoparticles (NPs) and Nisin, an antibacterial peptide, to form the Fe₃O₄-Phage-T4 + Nisin complex. The aim is to enhance antimicrobial efficacy and biofilm eradication. The Fe₃O₄-Phage-T4 + Nisin complex was synthesized by conjugating T4 bacteriophage with modified Fe₃O₄ NPs and Nisin. The antimicrobial and antibiofilm activity of the complex was evaluated against multidrug-resistant <i>Pseudomonas aeruginosa strains</i> (<i>PA01</i> and <i>PA14</i>) using biofilm inhibition and eradication assays. Stability and efficacy were further tested across a pH range of 5 to 8. The Fe₃O₄-Phage-T4 + Nisin complex exhibited superior biofilm removal compared to its individual components. The integration of Nisin broadened the antibacterial spectrum, targeting both <i>Gram-positive</i> and <i>Gram-negative bacteria</i>, while the modified Fe₃O₄ NPs enhanced phage penetration and bacterial cell disruption. The complex demonstrated significant biofilm inhibition and eradication, addressing the challenge of biofilm-related antibiotic tolerance, which often necessitates high antibiotic doses. Additionally, it maintained stability and efficacy across varying pH conditions.</p>