A combination of viral, bacterial, and host factors contribute to the severity and overall mortality associated with influenza virus:bacteria super-infections. To date, the virulence associated with the recently-identified influenza virus protein PB1-F2 has been largely defined using models of primary influenza virus infection, with only limited assessment in models of Streptococcus pneumoniae super-infection. Specifically, these studies have incorporated isogenic viruses that differ in the PB1-F2 expressed, but there is still knowledge to be gained from evaluation of natural variants derived from a non-human host species (swine). Using this rationale, we developed the hypothesis that naturally-occurring viruses expressing variants of genes, like PB1-F2, can be associated with the severity of secondary bacterial infections. To test this hypothesis, we selected viruses expressing variants in PB1-F2 and evaluated outcomes from super-infection with three distinct Gram-positive respiratory pathogens: Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes. Our results demonstrate that the amino acid residues 62L, 66S, 75R, 79R, and 82L, previously proposed as molecular signatures of PB1-F2 virulence for influenza viruses in the setting of bacterial super-infection, are broadly associated with enhanced pathogenicity in swine in a bacteria-specific manner. Furthermore, truncated PB1-F2 proteins can preferentially increase mortality when associated with S. pyogenes super-infection. These findings support efforts to increase influenza virus surveillance to consider viral genotypes that could be used to predict increased severity of super-infections with specific Gram-positive respiratory pathogens.