Clinical mastitis (CM) remains a critical challenge in dairy production, exacerbated by the global rise of antibiotic-resistant pathogens, which threatens herd health and productivity. This study pioneers a dual genomic-computational strategy to develop bacteriocin-based therapeutics-a promising alternative to conventional antibiotics-by targeting conserved virulence mechanisms in CM-causing pathogens. We aimed to (i) identify essential core proteins in CM-causing pathogens of dairy cows using the genomic approach; and (ii) assess the efficacy of bacteriocin peptides (BPs) as novel therapeutic agents targeting the selected core proteins for sustainable management of mastitis. Through pan-genomic analysis of 16 clinically relevant pathogens, including Staphylococcus aureus, S. warneri, Streptococcus agalactiae, S. uberis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, P. putida, and P. asiatica, we identified 65 evolutionarily conserved core proteins. Prioritization based on essentiality, virulence, and resistance potential revealed Rho (transcription termination factor) and HupB (nucleoid-associated protein) as high-value therapeutic targets due to their critical roles in bacterial survival and pathogenicity. A computational screen of 70 BPs identified 14 candidates with high binding affinity for both Rho and HupB proteins. Molecular dynamics simulations demonstrated that BP8, a novel dual-action bacteriocin, competitively inhibits Rho-mediated transcription termination and disrupts HupB-DNA interactions, effectively crippling bacterial replication and virulence. BP8 exhibited superior structural stability and binding efficacy compared to other candidates, positioning it as a potent broad-spectrum agent against diverse CM pathogens, including multidrug-resistant strains. Our study underscores the untapped potential of bacteriocins in veterinary medicine, offering a sustainable solution to mitigate antibiotic overuse and resistance. The computational validation of BP8 provides a foundational framework for developing targeted therapies, with implications for reducing dairy industry losses and improving animal welfare. Further in vitro and in vivo studies are warranted to translate these insights into practical therapeutics.