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There is widespread interest in using obligately lytic bacteriophages (“phages”) to treat human bacterial infections. Among <i>Staphylococcus aureus</i> infections, the USA300 lineage is a frequent cause of invasive disease. We observed that phage K, a model <i>S. aureus</i> myophage, exhibits temperature-sensitive growth on USA300 strains, with the wild-type phage providing poorer growth suppression in broth and forming smaller and fainter plaques at 37 °C vs. 30 °C. We isolated 65 mutants of phage K that had improved plaquing characteristics at 37 °C when compared to the parental phage. In all 65 mutants, this phenotype was attributable to loss-of-function (LoF) mutations in <i>gp102</i>, which encodes a protein of unknown function that has homologs only among the <i>Herelleviridae</i> (SPO1-like myophages infecting gram-positive bacteria). Additional experiments with representative mutants consistently showed that the temperature-sensitive plaque phenotype was specific to USA300 MRSA strains and that Gp102 disruption was correlated with improved suppression of bacterial growth in broth and improved antibacterial activity in a mouse model of upper respiratory tract infection. The same genotype and in vitro phenotypes could be replicated in close relatives of phage K. Gp102 disruption did not have a detectable effect on adsorption but did delay cell culture lysis relative to wild-type under permissive infection conditions, suggesting that <i>gp102</i> conservation might be maintained by selective pressure for more rapid replication. Expression of <i>gp102</i> on a plasmid was toxic to both an MSSA and a USA300 MRSA strain. Molecular modeling predicts a protein with two helix-turn-helix domains that displays some similarity to DNA-binding proteins such as transcription factors. While its function remains unclear, <i>gp102</i> is a conserved gene that is important to the infection process of <i>Kayvirus</i> phages, and it appears that the manner in which USA300 strains defend against them at 37 °C can be overcome by <i>gp102</i> LoF mutations.