Biofilm-associated polymicrobial infections, particularly those involving fungi and bacteria, are responsible for significant morbidity and mortality and tend to be challenging to treat. Candida albicans and Staphylococcus aureus are considered leading microbial pathogens primarily due to their ability to form biofilms on indwelling medical devices. However, the impact of mixed species biofilm growth on therapy remains largely understudied. Here, we demonstrate that in mixed biofilms, C. albicans confers S. aureus significantly enhanced tolerance to antimicrobials mediated by impairment of drug diffusion through the biofilm matrix composed of C. albicans secreted fungal cell wall polysaccharides. Further, we demonstrated a key role for the C. albicans secreted quorum sensing molecule farnesol in modulating S. aureus response to therapy via modulation of drug efflux pumps. Importantly, farnesol was also found to inhibit the production of the carotenoid pigment staphyloxanthin, an important virulence factor in S. aureus involved in protection against oxidative stress. The significance of this inhibition was established through demonstration of the enhanced susceptibility of the depigmented S. aureus cells to oxidants and macrophage phagocytic killing. Theoretical computational binding models indicated that the mechanism for pigment inhibition may be due to farnesol competitively inhibiting the binding of farnesyl diphosphate to CrtM, an essential enzyme in the biosynthesis of staphyloxanthin. To begin to explore the implications of this fungal-bacterial interactions in a host, we utilized a clinically relevant subcutaneous catheter mouse model where central venous catheter segments infected in vitro with S. aureus and C. albicans individually or in combination, are implanted subcutaneously in mice. Vancomycin treatment of mice with infected catheters demonstrated that where therapy significantly reduced S. aureus recovery from catheters in mice infected with S. aureus, in co-infected animals, vancomycin had no impact on S. aureus recovery, underscoring the therapeutic implications of mixed biofilm-associated infections. The combined findings from this work provide lacking mechanistic insights into interspecies interactions in biofilm with great clinical relevance. The understanding of the interspecies dynamics of signaling central to the persistence and antimicrobial resistance of polymicrobial infections will greatly aid in overcoming limitations of current therapies and in designing novel therapeutic strategies to target these complex infections. Therefore, future research should focus on designing animal model systems to study the role of secreted quorum sensing molecules in mediating these interactions in vivo, and the impact of these interactions on pathogenesis.