Candida albicans and Staphylococcus aureus form vigorous polymicrobial biofilms in serum, which may serve as the source of coinfection in patients. More importantly, S. aureus is highly resistant to vancomycin during polymicrobial biofilm formation, with no decreases in bacterial viability observed with up to 1,600 microg/ml drug. In these mixed-species biofilms, S. aureus preferentially associates with C. albicans hyphae, which express a variety of unique adhesins. We tested C. albicans mutants deficient in transcriptional regulators of morphogenesis (CPH1 and EFG1) and biofilm formation (BCR1) to investigate the role of hyphae in mediating polymicrobial biofilm formation. These mutants also have reduced expression of hypha-specific adhesins. The ability to form polymicrobial biofilms correlated with the ability to form hyphae in these mutants. However, only mutants that could adhere to the abiotic surface could induce S. aureus vancomycin resistance, regardless of the presence of hyphae. In examining factors that may mediate interspecies adhesion, we found that the C. albicans ALS family of adhesins (Als1 to Als7 and Als9) was not involved, and neither was the hypha-specific adhesin Hwp1. Therefore, polymicrobial biofilm formation and subsequent antibiotic resistance is a multifactorial process that may require a unique combination of fungal and/or bacterial adhesins.