Abstract During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model, which enables exploring the parameter space of the WT and DI competition. We also experimentally examined virus and DI replication kinetics during co-infection, and used these data to infer model parameters. Our model identifies, and our experimental measurements confirm, that the efficiencies of DI genome replication and encapsidation are two most critical parameters determining the outcome of WT replication. However, an equilibrium can be established which enables WT to replicate, albeit to reduced levels. Author summary We used a combination of mathematical modeling and experimental measurements to better understand the interaction between poliovirus (WT) and defective viral genomes (DI) co-infecting a single cell. We developed an ordinary differential equations (ODEs) model that capture the dynamics of the interaction, and provides a quantitative evaluation of the parameters, and their interactions, affecting virus replication. We observed that WT and DI compete directly for capsid proteins during encapsidation and for shared resources necessary for replication, a phenomenon known as exploitation competition. Hence, our study identifies two different types of competition occurring during the co-infection of a cell by WT and DI.