Abstract Staphylococcus aureus and Streptococcus pyogenes colonize mucosal surfaces of the human body to cause disease. A group of virulence factors known as superantigens are produced by both of these organisms that allows them to cause serious diseases from the vaginal (staphylococci) or oral mucosa (streptococci) of the body. Superantigens interact with T cells and APCs to cause massive cytokine release to mediate the symptoms collectively known as toxic shock syndrome. In this study we demonstrate that another group of virulence factors, cytolysins, aid in the penetration of superantigens across vaginal mucosa as a representative nonkeratinized stratified squamous epithelial surface. The staphylococcal cytolysin α-toxin and the streptococcal cytolysin streptolysin O enhanced penetration of toxic shock syndrome toxin-1 and streptococcal pyrogenic exotoxin A, respectively, across porcine vaginal mucosa in an ex vivo model of superantigen penetration. Upon histological examination, both cytolysins caused damage to the uppermost layers of the vaginal tissue. In vitro evidence using immortalized human vaginal epithelial cells demonstrated that although both superantigens were proinflammatory, only the staphylococcal cytolysin α-toxin induced a strong immune response from the cells. Streptolysin O damaged and killed the cells quickly, allowing only a small release of IL-1β. Two separate models of superantigen penetration are proposed: staphylococcal α-toxin induces a strong proinflammatory response from epithelial cells to disrupt the mucosa enough to allow for enhanced penetration of toxic shock syndrome toxin-1, whereas streptolysin O directly damages the mucosa to allow for penetration of streptococcal pyrogenic exotoxin A and possibly viable streptococci.