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Lactobacillus is a genus of gram-positive, facultative anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria. Lactobacillus acidophilus plays a most important role in the fermented products of the dairy industry and delivers the therapeutic characteristics of human health. As the greater quantity of antibiotics was used for the treatment of humans and farm animals as well and even for fish in aquaculture, that will result in the selection of pathogenic bacteria resistant to multiple drugs. Several research a great focus on the Identification of Lactic Acid Bacteria, that include conventional biochemical examinations like carbohydrate fermentation outlines using commercially accessible kits, physiological tests, and complex techniques by using molecular-based biology methods. More recently, Bio-log and API Kit have been applied to an enormous amount for Lactic Acid Bacteria Identification. The current study is carried out to isolate, identify, and evaluate the antagonistic activity of L. acidophilus. obtained from curd and milk samples as well. The antimicrobial action of this species was assessed with the agar well diffusion method against some pathogenic bacteria (Staphylococcus aureus, Escherichia coli, and Salmonella typhi). L. acidophilus. isolated and identified based on biochemical characterization as catalase-negative, motility negative, citrate negative indole negative, and fermentation of carbohydrates. In the present study, the total number of six isolates were detected as Lactobacillus sp. The isolated and identified L. acidophilus strains (n=6) were examined for their antagonistic activity against MDR strains of S. aureus, E. coli. and S. typhi.In the study, maximum inhibition was detected by LA1and LA4 against S.aureus (18mm) and S.typhi (18mm), respectively. The bare minimum inhibition was noticed in the case of LA3 against S. typhi i.e., 4.0mm. Other Lactobacillus isolates exhibited modest or nil antagonistic activity against tested pathogenic bacteria.