Abstract <p>Bovine respiratory disease (BRD) remains a major economic and animal health concern in cattle production, exacerbated by rising antimicrobial resistance and limited vaccine efficacy. Endolysins, phage-derived peptidoglycan hydrolases, have emerged as promising alternatives to antibiotics, but their activity against gram-negative bacteria is hindered by the outer membrane (OM) barrier. In this study, we identified five distinct prophage-derived endolysins from <i>Mannheimia haemolytica</i> and successfully expressed two (185 and 587AP2) along with their engineered polycationic nanopeptide&#xa0;(PCNP) fusions (PCNP-185 and PCNP-587AP2). All four recombinant proteins demonstrated muralytic activity, with original endolysins exhibiting higher enzymatic efficiency. However, PCNP-fused variants showed superior antibacterial effects as compared to those without. Especially, PCNP-185 achieved the most consistent multi-log₁₀ reductions of up to 4.5 in bacterial counts across <i>M. haemolytica</i> serotypes, particularly when combined with EDTA which increased permeability of the OM. These findings highlight the synergistic potential of engineered endolysins and EDTA in overcoming the gram-negative barrier. Additionally, selecting an appropriate buffer was found crucial to rule out the effect of buffers on antibacterial activity of endolysins. To our knowledge, this is the first report of engineering and evaluating prophage-derived endolysins against <i>M. haemolytica</i>, underscoring their promise as innovative therapeutics for BRD.</p> Key points <p>•&#xa0;<i>Prophage</i><i> endolysins are potent antibacterials with engineering potential.</i></p> <p>•&#xa0;<i>Chelator synergy boosts endolysin efficacy; buffer choice is critical.</i></p> <p>•&#xa0;<i>Endolysins hold strong promise as next-generation therapeutic agents against multi-drug-resistant M. </i><i>haemolytica causing bovine respiratory disease.</i> </p> Graphical abstract <p></p>