is a Gram-negative pathogen that causes a variety of infections in humans and animals. Due to the inappropriate use of antibiotics, multi-drug resistant (MDR) strains have emerged and are prevailing. In recent years, cow mastitis caused by MDR has attracted attention. In this study, a microbial community analysis revealed that could be a cause of pathogen-induced cow mastitis. Five MDR strains were isolated from milk diagnosed as mastitis positive. To seek an alternative antibacterial agent against MDR, , a lytic phage, designated vB_PaeS_PAJD-1 (PAJD-1), was isolated from dairy farm sewage. PAJD-1 was morphologically classified as and was estimated to be about 57.9 kb. Phage PAJD-1 showed broad host ranges and a strong lytic ability. A one-step growth curve analysis showed a relatively short latency period (20 min) and a relatively high burst size (223 PFU per infected cell). Phage PAJD-1 remained stable over wide temperature and pH ranges. Intramammary-administered PAJD-1 reduced bacterial concentrations and repaired mammary glands in mice with mastitis induced by MDR . Furthermore, the cell wall hydrolase (termed endolysin) from phage PAJD-1 exhibited a strong bacteriolytic and a wide antibacterial spectrum against MDR . These findings present phage PAJD-1 as a candidate for phagotherapy against MDR infection.