In recent years, gut-tailored probiotics have been proven to be beneficial for host health. Probiotic strains such as lactic acid bacteria (LAB) are known to exhibit antimicrobial activity, acting as natural substitutes for the regulation of foodborne pathogens. In the present study, a complete analysis, isolation, biochemical characterization, and molecular identification of Pediococcus acidilactici (NMCC-11) from Nili Ravi water buffalo (Bubalis bubalis) gut was carried out. NMCC-11 showed the best enzymatic potential, antimicrobial activity against known pathogenic strains, and survivability at a wide pH range (pH 4–pH 6) out of all isolates. The isolates were screened for their antimicrobial activity against the five most infectious microbes such as Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC9027), Staphylococcus aureus (ATCC6538), Listeria monocytogenes (ATCC13932), and Bacillus cereus (ATCC 11778) using the agar-well diffusion method. Moreover, after NMCC-11 isolation, a comparative diversity analysis against a variety of other randomly selected strains from around the world was carried out using R software. This study showed relatively low genetic diversity, which also contributed to the claim of the stability of this probiotic strain and its potential use as a starter culture and feed probiotic in the dairy industry. However, further studies are certainly warranted to determine its optimal dosage, time frame, and intake frequency.