| Summary: | In the escalating battle against antimicrobial resistance, there is an urgent need to discover and investigate new antibiotic strategies. Bacteriophages are untapped reservoirs of such potential antimicrobials. This study focused on Hypothetical Proteins of Unknown Function (HPUFs) from a <i>Staphylococcus</i> phage Stab21. We examined its HPUFs for bactericidal activity against <i>E. coli</i> using a Next Generation Sequencing (NGS)-based approach. Among the 96 HPUFs examined, 5 demonstrated cross-species toxicity towards <i>E. coli</i>, suggesting the presence of shared molecular targets between <i>E. coli</i> and <i>S. aureus</i>. One toxic antibacterial HPUF (toxHPUF) was found to share homology with a homing endonuclease. The implications of these findings are profound, particularly given the potential broad applicability of these bactericidal agents. This study confirms the efficacy of NGS in streamlining the screening process of toxHPUFs, contributes significantly to the ongoing exploration of phage biology, and offers promises in the search for potent antimicrobial agents.
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