Introduction: Probiotic activity of L. acidophilus and L. rhamnosus was great intend to treat pathogenic organism. The antibiotic resistance problem, it is important to develop Lactobacillus based approaches to combat Citrobacter koseri, Enterobacter cloacae, Serratia marcescens, Morganella morganii, and Proteus mirabilis, Proteus vulgaris and Klebsiella aerogenes-induced urinary tract infections, catheter-associated infections, and intestinal tract infections in the current situation. Aim/Objectives: In this study, our goal was to assess the in vitro antimicrobial activity of L. acidophilus and L. rhamnosus against clinical isolates of MDR, XDR, Non-MDR and ESBL strains of C. koseri, E. cloacae, S. marcescens, M. morganii, P. mirabilis, P. vulgaris and K. aerogenes. Methods: Antibiotic susceptibility of the test strains was determined by Kirby-Bauer disc diffusion method. The antimicrobial activities of treated and untreated L. acidophilus and L. rhamnosus against the above mentioned MDR, XDR, Non-MDR and ESBL pathogens were determined by agar overlay spot test. Mean zone of inhibition was statistically analyzed using ANOVA single factor. Results: Among L. acidophilus and L. rhamnosus the mean value displayed the potent activity against most test isolates with mean zones of inhibition ranging from 4 to 15 mm and the p-value