The extensive use of antibiotics in animal feed and development of antimicrobial resistance has led to the development of probiotics to improve the rate of livestock productivity. The present study was designed to isolate breed specific lactic acid bacterial (LAB) strains as animal probiotics. Sixty-eight gram-positive and catalase-negative strains were isolated, from the gastrointestinal tract and milk of Nilli Ravi buffaloes. For the selection of the buffalo probiotic, the viable capability of the isolated strains in a medium with different rumen conditions and antimicrobial activity were determined. Six strains (NMCC- PI, NMCC- PT, NMCC- P3n, NMCC- P7, NMCC- P8 and NMCC- F) showed a positive antibacterial as well as maximum cellular viability. These strains were identified as Enterococcus lactis (NMCC- PI) Pediococcus pentosaceus (NMCC- PT, MK014295), Enterococcus faecalis (NMCC-P3n) Lactobacillus fermentum (NMCC-P7), Pediococcus acidilactici (NMCC-P8) and Enterococcus ratti (NMCC-F) through 16 S rRNA gene sequencing. All strains fulfilled the basic criteria of animal probiotic properties however P. acidilactici (NMCC-P8) showed best result in terms of survivability at low pH (2.0), better antimicrobial activity and maximum enzymatic potential. Present results demonstrated that P. acidilactici may serve as a promising candidate probiotic for use in the buffalo dairy industry. This study was the unique to select LAB, with the potential to be used as probiotic from Nilli Ravi buffalo ruminal gut based on the ruminal-gut conditions.