Probiotics are living microbial adjuncts which has valuable effects on host body incuding improved food digestion, enhancing host‟s immune responses, modification of host associated microbial community and thus, improving the overall ambient environment. Since increase in multidrug resistance pathogens makes the standard treatments in-effective, probiotic have been developed sccessfully as alternate therapeutic drugs in recent era. Commercialized synthetic drugs (e.g. sulfa drugs) have also been interlinked with severe side effects to the hosts which have ultimately lemmatized their use. Bacteriocin (peptide antimicrobial) producing bacteria that can competitively exclude pathogens in the host, is an emrging approach towards novel therapeutics. These defensive proteins are released by 95% of bacterial species in their natural habitat to compete with one another for food and space. Purified bacteriocins of Lactobactericeae (lantibiotics) have been commercialized as prebiotics after successful experiments in laboratory animal. Bacteriocins of Gram negative bacteria (colicins and microcins) are also able to competitively exclude closely related bacterial species when fed to model organism and livestock. The current study was carried out to isolate commensal Gram negative strains that can hinder the growth of common pathogens by producing colicins and microcins against them. Commensal E.coli strains from cattle, sheep and humans were the choice of study since they were expected to better adapt to the host body when administrated as probiotics. Phenotypic inhibitory activity was determined, followed by genomic detection of various colicins, microcins and virulence determinants among the active isolates. Moreover, these strains were also evaluated for antibiotic sensitivity profile, pH tolerance, hemolytic activity and entero-invasive property. The E.coli strains were also sorted into phylogenetic groups of E.coli i.e. A0, A1, B1, B2, D1 and D2. From 300 fecal samples of cattle, sheep and human, 675 lactose fermenting Gram negative colonies were selected, out of which 513 were confirmed as E.coli based on biochemical characterization. For accuracy of further tests, 465 E.coli isolates (n=155/ sample) were evaluated in vitro for growth inhibition of five Gram negative species i.e. E.coli O157:H7, E.coli O26:H11, Salmonella enterica, Klebsiella pneumonia and Pseudomonas aeruginosa. Only 68 isolates (26.4% cattle, 25% sheep and 48.5% human) produced significant growth inhibition among which 41.1% inhibited E.coli O157:H7, 35.2% E.coli O26:H11, 30.7% S. enterica, 24.8% P. aeruginosa and 13% inhibited K. pneumonia. Genomic enumeration of colicin and microcin determinants of these antagonistic E.coli isolates revealed that each of the isolate produced at-least one colicin while the microcins were rarely found in commensal species. The highest prevailing colicin was col E6 (70.4%) followed by col Ib (66.3%), E4 (54.28%), E7 (49.9%), col J (42.9%), col M (35.2%) and col Ia (29.3%). Colicins like S4, D, A, E1, E3 and microcins including mH47, mV, mB17 and mC7 were prevalent at intermediate levels (< 25%) while col E2, 10 and mB17 were rarely found (< 5%) in E.coli isolates. Determinants for col B, K, 5 and mL were not perceived in any E.coli isolate. Phylogenetic grouping revealed that group B2 E.coli was more prevelant (i.e. 27.9%) among human samples followed by 13.2% in sheep and 11.7% in cattle while group D1 was the second most prevalent group i.e. 16%. Almost 13.2 % B1 E.coli, 11.6% A1 and 5.8% A0 E.coli were found among the antagonistic strains from all sources. Virulence gene determinants (i.e. Stx1, Stx2, Hlye, St and eaeA) were detected in 60.2% E.coli isolates while remaining 39.7% were non-pathogenic commensal isolates which were further confirmed by 16SrRNA sequencing. Antibiotic susceptibility profile, hemolysis assay, entero-invasive ability and pH tolerance of all non-pathogenic strains revealed that out of total E.coli strains, four cattle, two human and one sheep derived E.coli strains were the most suitable strains to be considered as probiotic species. However, further insight is suggested to unveil the effects of downstream processes and to discover eminent capsule packaging for these strains so that they can be easily fed to hosts.