The aim of this study was to isolate and characterize bovine-vaginal probiotics genotypically and phenotypically usingin silicoand evaluate theirin vivoperformance in buffaloes with endometritis. For thein vitroisolation and characterization, vaginal swabs were collected from 34 cows and 17 buffaloes, and 709 primary bacterial isolates with probiotic activity were obtained using MRS agar media. Two isolatesLactiplantibacillus plantarumKUGBRC (LPKUGBRC) andPediococcus pentosaceusGBRCKU (PPGBRCKU) demonstrated optimumin vitroprobiotic activities as compared toLacticaseibacillus rhamnosusGG including, acid production, secretion of fatty acids and exopolysaccharide, cell surface hydrophobicity, self-aggregating and co-aggregating capacity with pathogens, anti-microbial activity and bacteriocin-like compounds against pathogensEscherichia coliandStaphylococcus aureusin cell-free supernatant and absence of hemolytic activity. Their phenotypic capacity was confirmed by analyzing the whole genome sequencing data and identifying genes and pathways associated with probiotic properties. These probiotic isolates have shown no virulence genes were discovered in their genomic study.In vivostudy of 92 buffaloes suffering from clinical endometritis with purulent cervico-vaginal mucus (CVM) were randomly allocated 40 × 108CFU/ml LPKUGBRC and PPGBRCKU and 40 ml Normal saline. The LPKUGBRC reduced the duration between administration of probiotic to induction of healthy estrus significantly. However, no effect was observed on pregnancy rate. These results suggest that LPKUGBRC and PPGBRCKU probiotic bacteria demonstrate probiotic efficiency and adaptability. Further sourced from the same niche as the targeted infection, they offer a distinct advantage in targeting the specific microbial population associated with endometritis. The findings of this study highlight the potential of LPKUGBRC and PPGBRCKU probiotics in treating endometritis and suggest further exploration of their clinical applications.