BACKGROUND: Tumor-associated macrophages (TAMs) constitute an important part of the tumor microenvironment of breast cancer (BC), and they play an essential role in modulating tumor growth and invasion. However, the role of TAMs in neoadjuvant chemotherapy (NAC) has not been fully elucidated. Therefore, the aim of this study was to assess the function of TAM subtypes and investigate their role in the response to NAC in BC. METHODS: Presence of TAMs was examined immunohistochemically (IHC) in pre- and post- NAC treatment tumor tissue in a cohort of 138 BC patients. IHC staining with monoclonal antibodies for CD68 and CD163 were performed. Positivity was defined as staining > 1% TAMs in stroma and tumor cell nests. Response to NAC was evaluated according to tumor size change and Residual Cancer Burden (RCB) index. RESULTS: CD68+ and CD163+ TAMs decreased significantly in both the stroma and tumor nests (TN) after NAC. The median CD68+ TAMs in the stroma decreased significantly from 5% to 1% (p < 0.005), while CD163+ TAMs showed a marked reduction from 20% to 5% (p < 0.001). Post-NAC, the persistence of CD68+ and CD163+ TAMs in the stroma was strongly correlated with larger residual tumor size (p < 0.005 and p < 0.001, respectively). Changes in CD163+ TAM levels in the stroma were significantly associated with RCB classes (p < 0.005). Pre-NAC, CD163+ TAMs in the stroma and TN showed a significant association with TILs; however, no correlations with TILs were observed post-NAC. CONCLUSION: This study highlights the critical role of TAMs dynamics in shaping NAC response in BC. Notably, CD163+ TAMs may emerge as pivotal players in mechanisms of chemotherapy resistance and response, underscoring their potential as biomarkers and therapeutic targets in breast cancer treatment.