Staphylococcus aureus is the most common multidrug resistant organism isolated from nosocomial infections. With the reduced approval and availability of effective antibiotics, novel treatment protocols for this pathogen are needed. Furthermore, biofilms display an inherent resistance to antibiotics and are associated with the majority of chronic diseases in humans. Therefore, we sought to evaluate the effectiveness of hydrolytic enzymes secreted by a host-independent variant of Bdellovibrio bacteriovorus on S. aureus and its biofilms. LC-MS analysis identified numerous secreted proteases between 30 and 70 kDa in size. Addition of this supernatant (10%) to growing cultures or pre-formed biofilms of S. aureus led to a significant loss (70~80%) in its ability to form or maintain a biofilm. Addition of protease inhibitors, such as AEBSF, demonstrated that serine proteases contributed to this loss. Although the growth of S. aureus was not altered by addition of the supernatant, comparative SDS-PAGE analysis of its surface proteins found that many were significantly lower after treatment. Furthermore, the supernatant had no effect on the viability of MCF-10a human epithelial cells but did reduce S. aureus invasion into these cells by 5-fold. As such, this study demonstrates the hitherto unknown capability of B. bacteriovorus to mitigate gram positive biofilms and their virulence, furthering the potential application of this strain as a probiotic and in the treatment of diseases.