[Objective] To identify the main pathogenic bacteria causing clinical mastitis in dairy cows at a large-scale farm in Xinjiang and determine their antibiotic resistance profiles,providing a scientific basis for targeted prevention and control strategies. [Method] Aseptically collected milk samples from 15 cows with clinical mastitis were subjected to bacterial isolation and purification,morphological observation,physiological and biochemical identification,and 16S rRNA molecular characterization. The antibiotic susceptibility of the isolated strains was assessed using the Kirby-Bauer （K-B） disk diffusion method against 12 commonly used antibiotics. [Result] A total of 21 bacterial strains were isolated,including Escherichia coli （11 strains,52.38%）,Staphylococcus aureus （7 strains,33.33%）,and Streptococcus spp. （3 strains,14.28%）. Antibiotic susceptibility testing revealed that E. coli exhibited complete resistance to amoxicillin and clindamycin but moderate sensitivity to gentamicin and enrofloxacin. S. aureus was resistant to polymyxin but highly sensitive to most other antibiotics. Streptococcus spp. showed high sensitivity to tetracycline and enrofloxacin. Mixed bacterial susceptibility testing indicated that enrofloxacin,ofloxacin,and tetracycline were the most effective treatments. [Conclusion] Clinical mastitis in this farm was primarily caused by mixed infections of E. coli,S. aureus,and Streptococcus spp. The recommended treatment regimen includes enrofloxacin,ofloxacin,and tetracycline,complemented by comprehensive measures such as regular screening for subclinical mastitis,environmental management improvements,and strict disinfection protocols to effectively control the occurrence and spread of mastitis.