S. aureus biofilm infections, common in patients with chronic wounds or artificial implants, are clinically significant due to their ability to subvert the human innate immune response and to result in persistent illness. Previous investigations suggest that S. aureus biofilms attenuate macrophage-mediated antimicrobial function by disrupting NF-κB-coordinated transcription of proinflammatory intermediates. However, the identity of one or more bacterial molecules responsible for this dysfunction remains unknown. This study investigates the S. aureus-produced protein alpha toxin as a potential disruptor of the complex signaling process that leads to inflammation. In this study, antibacterial receptor agonists for TLR2, TLR9, and NOD2 are used to synthetically stimulate RAW 264.7 macrophages toward an M1 pro-inflammatory phenotype while they are co-cultured in conditioned medium from cultured wild-type or alpha toxin-knockout S. aureus biofilms. Using qPCR analysis and an NF-kB reporter assay, the pro-inflammatory response of macrophages in either environment can be quantified and compared to assess the effect of alpha toxin on one or more receptor-mediated pathways. Characterization of this host-microbe interaction can lead to a better understanding of the innate immune response, as well as to the development of targeted therapies to improve healthcare outcomes for patients with S. aureus biofilm infections.