Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature monocytes and granulocytes that are potent inhibitors of T cell activation. A role for MDSCs in bacterial infections has only recently emerged, and nothing is known about MDSC function in the context of Staphylococcus aureus infection. Because S. aureus biofilms are capable of subverting immune-mediated clearance, we examined whether MDSCs could play a role in this process. CD11b+ Gr-1+ MDSCs represented the main cellular infiltrate during S. aureus orthopedic biofilm infection, accounting for> 75% of the CD45+ population. Biofilm-associated MDSCs inhibited T cell proliferation and cytokine production, which correlated with a paucity of T cell infiltrates at the infection site. Analysis of FACS-purified MDSCs recovered from S. aureus biofilms revealed increased arginase-1, inducible NO synthase, and IL-10 expression, key mediators of MDSC suppressive activity. Targeted depletion of MDSCs and neutrophils using the mAb 1A8 (anti-Ly6G) improved bacterial clearance by enhancing the intrinsic proinflammatory attributes of infiltrating monocytes and macrophages. Furthermore, the ability of monocytes/macrophages to promote biofilm clearance in the absence of MDSC action was revealed with RB6-C85 (anti–Gr-1 or anti-Ly6G/Ly6C) administration, which resulted in significantly increased S. aureus burdens both locally and in the periphery, because effector Ly 6C monocytes and, by extension, mature macrophages were also depleted. Collectively, these results demonstrate that MDSCs are key contributors to the chronicity of S. aureus biofilm infection, as their immunosuppressive function prevents monocyte/macrophage proinflammatory activity, which facilitates biofilm persistence.