In nature, microbes rarely grow as mono-species planktonic forms. Rather, most are associated as polymicrobial biofilms attached to host and environmental surfaces. The polymorphic fungus Candida albicans and bacterium Staphylococcus aureus are both capable of forming biofilms, can be co-isolated from virtually all human mucosal sites, and are responsible for diverse localized and deep-seated infections. Despite causing a large number of mono-species infections, they have been implicated as co-infecting organisms in a variety of human diseases. Therefore, various approaches were used to understand the intricacies of this medically relevant dual-species biofilm. Confocal scanning laser microscopy and species specific PNA-FISH analysis revealed that S. aureus possesses an affinity for the pathogenic hyphal form of C. albicans in vitro. C. albicans hyphae are directly invasive and have been demonstrated previously to penetrate host epithelial tissue; therefore, we hypothesized that S. aureus may use the hyphae of C. albicans to become invasive. An in vitro co-infection of human keratinized epithelium demonstrated the ability of S. aureus to attach to C. albicans hyphae and penetrate the epithelial cells, while cells infected with S. aureus alone showed no such breach of the epithelial surface. A similar invasive pattern was also demonstrated during infection of explanted murine tongue tissue. An in vivo mouse model of oral co-infection established the ability of S. aureus to translocate to the kidneys only in the presence of C. albicans while mono-species infections were either cleared or remained localized. Furthermore, through the use of various C. albicans cell wall protein knockout mutants and recombinant protein expression, we have demonstrated that staphylococcal hyphal binding is partially dependent on the candidal adhesin Als3p. We have also utilized differential in gel electrophoresis (DIGE) as a tool to partially define shifts in global protein expression due specifically to polymicrobial biofilm growth. Therefore, we present a novel mechanism of invasive S. aureus infection facilitated via interactions with C. albicans in an oral polymicrobial biofilm setting. These findings have significant impact on the consideration of treatment options for those afflicted with complicated biofilm mediated infections.