Simple SummaryMastitis is known to pose a public health hazard and cause a costly disease for dairy herds. Bacteria are the primary causes of mastitis; and antibiotics are widely used in the treatment of the disease. This can lead to antibiotic residues in various kinds of milk and an increase in the risk of antibiotic resistance in bacteria. To avoid these problems, alternative treatments by the use of natural products are being experimented and plants extracts are also examined. This study reports the investigation of potential antioxidants and antibacterial activities against mastitis pathogens of pomegranate flowers’ extracts. Results showed a high antioxidant and antibacterial potential of pomegranate flowers’ extracts against the examined mastitis pathogens.AbstractMastitis is one of the most considerable and costly diseases for dairy herds, andStaphylococcusspp. is known to be the main causative agent. Although antibiotics are widely used in the treatment of mastitis, this can cause both antibiotic residues in milk and the risk of antibiotic resistance occurrence in bacteria. Thus, in recent years, researchers have focused on alternative treatments for this disease and plants extracts are investigated for this purpose. Pomegranate is widely used as a dye, ornament, and medicinal plants in the industry, and the species has a particularly high economic value in Turkey. This study aims to investigate in vitro the antioxidant and antibacterial properties of the pomegranate flower’s extracts against differentStaphylococcusspecies associated with bovine mastitis. To this aim, pomegranate flowers were collected from different regions in Turkey and extracts were prepared with three different solvents (methanol, ethanol, and water). The retention factor values of the ethanol extract were determined by thin-layer chromatography. The antibacterial activity tests were carried out via the disk diffusion method. In addition, the extracts were tested against the stable DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radicals for antioxidant activity. Four retention factors (0.79, 0.67, 0.58, and 0.33 points) were found for the ethanol extract. The methanol extract showed the highest inhibition zones against coagulase-negativeStaphylococcus-37 (CNS-37) andS. aureus-18. The lowest MIC was 6500 μg/mL. The highest antioxidant activity was observed in methanol extracts. As a result, the extracts of pomegranate flowers showed a high antioxidant and antibacterial potential against the examined mastitis pathogens.