The emergence of antibiotic-resistant biofilm producing microorganisms such as Pseudomonas aeruginosa has pushed efforts to find safe alternatives to antibiotics; such as probiotics. Lactobacilli are one of these promising probiotics, with reported antibacterial and anti-biofilm activity against many different pathogenic microorganisms. This study aimed to study the potential antibacterial and anti-biofilm effect of Lactobacillus acidophilus ATCC 4356, against the growth and biofilm formation of pathogenic P. aeruginosa. Cell free supernatant of L. acidophilus was tested to inhibit the growth; biofilm formation, and on preformed biofilms by 35 different clinical strains of P. aeruginosa, using agar well diffusion and microtitre plate assays. L. acidophilus ATCC 4356 recorded powerful growth inhibition against 88.6% of the P. aeruginosa strains. Moreover; it significantly inhibited biofilm formation of the strains by 68.52%, and removed already preformed biofilms with 43.8 % activity. Finally; L. acidophilus showed a potent inhibitory potential against the growth and biofilm formation by P. aeruginosa strains, thus could be used as a powerful probiotic for the bio-control of infections caused by antibiotic resistant and biofilm producing P. aeruginosa.