Background & Objectives: Probiotics exert a strong antagonistic activity against many microorganisms including food spoilage organisms and enteropathogens. Lactobacilli find increasing acceptance as probiotics by showing its beneficial effects. Keeping in view the benefits of probiotics in literature, the present study was planned to isolate Lactobacillus spp. from different fermented dairy products and to study the inhibitory potential against biofilm forming bacteria viz. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Methods: Lactobacillus spp. were isolated from different fermented dairy products and characterized. The antimicrobial activity of each Lactobacillus spp. was checked by agar well diffusion and overlay methods. The strains showing inhibitions in these assays were further used to detect biofilminhibition against E. coli and S. aureus by microtiter plate biofilm assay method. Results: A total of 21 samples were studied of which pure growth was observed in 67% and mixed growth was observed in 33% of the samples. Of 18 isolates obtained, 45% belonged to Lactobacillus plantarum (L. plantarum), 33% were Lactobacillus acidophilus (L. acidophilus) and 22% comprised of Lactobacillus fermentum (L. fermentum). Ten isolates showed inhibition against E. coli and 9 isolates showed inhibitory activity against S. aureus in agar well diffusion assay and overlay method. In microtiter plate biofilm assay, the absorbance values were less in the wells where E. coli and S. aureus were mixed with the Lactobacillus broth. Interpretation and Conclusions: The Lactobacillus strains showing inhibitory activities against the pathogens can be used as probiotics or as starter culture in food fermentations after confirming other attributes.