Lactic acid bacteria (LAB) are applied as probiotics that stimulate digestion, immunity and represents suitable alternatives to conventional antimicrobials in animal production systems. The present study aimed to isolate, characterize, and examine the prospective LAB from Murrah buffalo calves' faecal samples as potential species-specific probiotics. Accordingly, 96 lactobacilli strains were isolated; out of which 55 isolates were Gram-positive, catalase-negative and vancomycin-resistant. These isolates had been presumptively identified as Lactobacillus species and further confirmed by genus-specific PCR. Seventeen isolates were shortlisted based on cell surface hydrophobicity (CSH) and auto-aggregation properties and identified by 16S rDNA sequencing. Cluster analysis from the phylogenetic tree revealed four different groups comprising of L. reuteri (11), L. salivarius (4), L. mucosae (1) and L. agilis (1). Out of these, seven isolates were non-hemolytic and showed better resistance to adverse gastrointestinal tract (GIT) conditions viz. tolerance to pH, bile salts and phenol. The selected isolates also exhibited significant (P