Neutrophils are vital components of the innate immune system, playing crucial roles in defending against pathogens and contributing to the pathogenesis of various diseases, including cancer and autoimmune disorders. Their functions, such as reactive oxygen species (ROS) production, phagocytosis, and microbial killing, are essential for immune responses, but excessive activation can lead to tissue damage. The influence of conditioned media (CM), derived from different cell types, on neutrophil function has gained significant attention. This study investigates the impact of human umbilical vein endothelial cell (HUVEC) derived CM (H-CM) on neutrophil activity. HUVECs were cultured in serum-free media to collect CM, which was then used to treat isolated neutrophils. The effects of H-CM on neutrophil viability, ROS production, and yeast-killing ability were assessed. Results showed that H-CM significantly reduced neutrophil viability, suppressed ROS production, and diminished the ability of neutrophils to kill yeast. These findings suggest that H-CM modulates neutrophil function by impairing key antimicrobial activities, which could be useful for regulating immune responses in diseases characterized by neutrophil dysregulation, such as autoimmune diseases and chronic inflammation. Further research is needed to explore the broader effects of H-CM on neutrophil functions and its potential therapeutic applications.