Background: Acinetobacter baumannii and Enterocoocus faecalis increase their resistance against antibiotic by producing biofilm. Antibiotic resistance has become a massive public health threat that require novel effective antibacterial and antibiofilm alternatives. The use of probiotics is interested to prevent and control certain infections. The objective of this study was to investigate the antibacterial and antibiofilm property of probiotics and synbiotics against multidrug-resistant A. baumannii and E. faecalis. Methods: The antimicrobial and the antibiofilm activities of cell- free supernatants of four strains of Lactobacillus against 20 clinical multi-drug resistant (MDR) isolates of Acinetobacter baumannii and Enterocoocus faecalis were determined in the presence of 0.3% of sorbitol, raffinose, citrate, trehalose, inulin, and riboflavin using well diffusion agar and micro-dilution method. Results: The cell- free supernatant of L. rhamnosus with citrate and trehalose showed the best antibacterial activity against MDR A. baumannii (28.8±2.1mm, 1.128 μL/mL), and L. rhamnosus with all of prebiotics against MDR E. faecalis (29.8±0 mm, 1.128 μL/mL) compare to probiotic alone. The prebiotics could improve the inhibitory effect of probiotics against the Gram-negative A. baumannii higher than Gram-positive E. faecalis. Biofilm formation was reduced in both pathogens in presence of synbiotics. L. plantarum with riboflavin and L. rhamnosus with or without inulin potently inhibits E. faecalis (50±0.86%) and A. baumannii (75±6.5%) biofilm formation, respectively. Conclusions: The results of current study support the antibiofilm activity of metabolites produced by synbiotics, and suggest their use as suitable adjuvants as well as biocontrol agents for treatment.