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B7-H6 and PD-L1 belong to the B7 family co-stimulatory molecules fine-tuning the immune response. The present work investigates the clinical effect of B7-H6 protein expression with PD-L1 status and the infiltration of natural killer cells as potential biomarkers in breast tumor inflammatory microenvironment. The expression levels of B7-H6 protein by cancer cells and immune infiltrating cells in human breast cancer tissues and evaluate their associations with PD-L1 expression, NK cell status, clinical pathological features and prognosis were explored. The immunohistochemistry labeling method was used to assess B7-H6 and PD-L1 proteins expression by cancer and immune cells. The associations between immune checkpoint, major clinical pathological variables and survival rates were analyzed. B7-H6 protein was depicted in both breast and immune cells. Results showed that Tumor B7-H6 expression is highly associated with Her-2 over expression. B7-H6 + immune cells are highly related to the Scarff–Bloom–Richardson grade and associated with PD-L1 expression and NK cells status. Survival analysis revealed a better prognosis in patients with low expression of B7-H6 by cancer cells. Conversely, B7-H6 + immune cells were significantly associated with longer survival. Findings strongly suggest an interaction between B7 molecules that contributes to a particular design of the inflammatory microenvironment. This may influence the efficiency of therapies based on antibodies blocking the PD-L1/PD1 pathway and can explain the detection of clinical benefits only in a fraction of patients treated with immune checkpoint inhibitors.