Background: Emergence of biofilm-producing P. mirabilis, especially those resistant to conventional antibiotics, necessitates safe and natural alternatives like probiotics. We investigated the potential antimicrobial and anti-biofilm activities of L. acidophilus and L. reuteri against P. mirabilis clinical isolates. Methodology: P. mirabilis colonies were identified by traditional methods. Antimicrobial susceptibility and biofilm formation were assessed by disk diffusion and microtiter plate assay (MTP) respectively. L. acidophilus and L. reuteri were incubated in De Man, Rogosa and Sharpe broth, then centrifuged to get cell free supernatant. Antimicrobial and anti-biofilm effect of supernatant against P. mirabilis were tested using agar well diffusion and MTP methods, respectively. Results: About 61.3% of P. mirabilis isolates were biofilm producers with 92% and 30.7% for ampicillin and imipenem resistance, respectively. Multi-drug resistance represented 42.7%. Supernatants of L. acidophilus and L. reuteri had 100% antimicrobial inhibition for untreated and 56% and 68% respectively for treated one with mean inhibition zone diameters 18.65±1.05 and 18.32±1.08 respectively for untreated and 8.44±7.54 and 10.0±6.93 respectively for treated supernatant. L. acidophilus supernatant showed significant reduction of biofilm formation by ≥78% and ≥75% for L. reuteri. Treated and untreated supernatant showed significant eradication of maturely formed biofilm by 37% - 70% for L. acidophilus and 40%- 66% respectively for L. reuteri. Conclusion: L. acidophilus and L. reuteri demonstrated antibacterial and anti-biofilm activity that with further in vivo and in vitro testing can be employed as a novel antimicrobial agent against P. mirabilis.