Breast cancer, the most prevalent malignancy in women, is influenced by the tumor microenvironment (TME), where interactions between cancer cells and immune cells, including neutrophils, play a significant role in tumor progression. Neutrophils, which can have anti-tumor (N1) and pro-tumor (N2) functions, are involved in immune responses such as reactive oxygen species (ROS) production. However, the effects of tumor-derived conditioned media (CM) on neutrophil function remain poorly understood. This study investigates the impact of 4T1-derived CM (4T1-CM) on neutrophil activity, focusing on ROS production and antimicrobial function. We isolated neutrophils from healthy mice and exposed them to 4T1-CM. Our results show that 4T1-CM significantly enhances ROS production, as demonstrated by increased nitroblue tetrazolium (NBT) reduction, and improves neutrophil-mediated yeast killing. Importantly, 4T1-CM did not affect neutrophil viability, as assessed by MTT assay. These findings suggest that 4T1-CM enhances neutrophil function, specifically their ROS production and antimicrobial activity, without compromising cell health. This study highlights the potential role of CM in modulating immune cell responses in the TME, with implications for breast cancer immunotherapy strategies.