Influenza A viruses (IAV) can undergo rapid evolution by acquisition of new genes through reassortment between IAV strains leading to immune evasion, antiviral resistance, and change in host range. Understanding of the factors that can enhance or reduce reassortment frequency therefore has implications for protection of individual and public health. Reassortment requires co-infection by two or more viral particles to the same host cell. Prior studies had identified the potential for IAV particles to aggregate on the bacterial surface of Streptococcus pneumoniae and other respiratory pathobionts, suggesting that bacterial cells could serve as a method to aggregate IAV particles and potentially facilitate reassortment. To test this hypothesis, a temperature sensitive, oseltamivir resistant viral strain was generated and used in in vivo and in vitro co-infection experiments with wild type virus in the presence of live and killed bacterial cells. While killed pneumococcal cells can enhance IAV reassortment frequency in a density-dependent manner, live pneumococci cannot, suggesting a product produced by viable pneumococci inhibits viral reassortment. Genetic deletion of the pneumococcal cholesterol-dependent cytolysin, Pneumolysin (Ply), in combination with inhibition of inflammatory signaling induced by Ply, restored the enhancement of reassortment to the levels seen with killed pneumococci. The results of this work suggest that the bacterial cells that colonize the human upper respiratory tract can have a complex role in modulating IAV reassortment frequency. Bacterial cells are capable of facilitating enhancement of viral reassortment, however, bacterial products can negate these effects. This work demonstrates the role of one such interaction with Ply inhibiting the enh