Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature monocytes and granulocytes. While neutrophils (polymorphonuclear leukocytes [PMNs]) are classically identified as highly differentiated cells specialized for antimicrobial defense, our laboratory has reported minor contributions of PMNs to the immune response during Staphylococcus aureus biofilm infection. However, these two cell types can be difficult to differentiate because of shared surface marker expression. Here we describe a more refined approach to distinguish MDSCs from PMNs utilizing the integrin receptor CD11b combined with conventional Ly6G and Ly6C expression. This approach separated the Ly6G⁺ Ly6C⁺ population that we previously identified in a mouse model of S. aureus orthopedic implant infection into two subsets, namely, CD11b^high Ly6G⁺ Ly6C⁺ MDSCs and CD11b^low Ly6G⁺ Ly6C⁺ PMNs, which was confirmed by characteristic nuclear morphology using cytospins. CD11b^high Ly6G⁺ Ly6C⁺ MDSCs suppressed T cell proliferation throughout the 28-day infection period, whereas CD11b^low Ly6G⁺ Ly6C⁺ PMNs had no effect early (day 3 postinfection), although this population acquired suppressive activity at later stages of biofilm development. To further highlight the distinctions between biofilm-associated MDSCs and PMNs versus monocytes, transcriptional profiles were compared by transcriptome sequencing (RNA-Seq). A total of 6,466 genes were significantly differentially expressed in MDSCs versus monocytes, whereas only 297 genes were significantly different between MDSCs and PMNs. A number of genes implicated in cell cycle regulation were identified, and in vivo ethynyldeoxyuridine (EdU) labeling revealed that approximately 50% of MDSCs proliferated locally at the site of S. aureus biofilm infection. Based on their similar transcriptomic profiles to those of PMNs, biofilm-associated MDSCs are of a granulocytic lineage and can be classified as granulocytic MDSCs (G-MDSCs).