The current study aimed to investigate the role of melatonin, B-carotene, selenium, and hydrocortisone in protecting a primary mammary cell line, as a model of mastitis, from the deleterious effects of reactive oxygen species (ROS) secreted from activated neutrophils in vitro. Different concentrations of melatonin (0.5, 5, 50, and 500 UM), B-carotene (1, 10, and 100 uM), selenium (0.001, 0.01, and 0.1 uM), and hydrocortisone (0.1, 1, and 10 uM) were added separately in triplicates to primary mammary epithelial cell lines prepared from active mammary tissue of female buffalo co-incubated with activated buffalo neutrophils (5 X 10/well). Mammary cell damage was evaluated by measuring lactate dehydrogenase (LDH), malondialdehyde (MDA) and nitric oxide (NO) concentrations in culture media as indicative of ROS, and by morphological observations of neutrophils after acridine orange staining. Activated neutrophils damaged the mammary epithelial cell line as demonstrated by an increase in LDH, MDA, and NO concentrations in control mammary cells that did not receive any treatments. Meanwhile, treated mammary epithelial cells with melatonin, B-carotene, selenium, and hydrocortisone secreted, considerably, low concentrations of LDH, MDA, and NO. Also photographs of the neutrophils treated with antioxidants or hydrocortisone did not show much damage as compared with non-treated control cells. Our results suggest that ROS such as superoxides, hydrogen peroxide, hydroxyl radical, and nitric oxide released by activated neutrophils cause damage to mammary epithelial cells indicated by increased concentrations of LDH, MDA, and NO and this deleterious effect could be quenched by the use of antioxidants su as melatonin, B-carotene, and selenium or by the use of anti inflammatory compound such as hydrocortisone