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Mastitis is one of the major prevailing and costly diseases in dairy cattle and buffaloes. This study describes the current prevalence of mastitis in lactating cattle, and buffaloes in Khyber Pakhtunkhwa during 2019-2020, to identify bacterial pathogens of mastitis, and to determine the antimicrobial resistance. A total of 1013 milk samples belonging to 726 cattle and 287 buffaloes were examined to detect clinical mastitis (CM) and subclinical mastitis (SCM). Mastitic milk samples (n=541) were further used to investigate the prevalence of bacteria associated with mastitis and their antimicrobial susceptibilities by standard disc diffusion assay. The results showed that the prevalence of CM and SCM was 21.2% and 62.3% in cattle, while 17.8% and 68.9% in buffaloes, respectively. Additionally, it was noted that CM was higher in cattle, while SCM was higher in buffaloes. Amongst the bacterial isolates from mastitic milk the prevalence of Staphylococci spp. (24.6%) was at highest, which was followed by Escherichia coli (23.7%), then Streptococci spp. (8%) Salmonella spp. (5.9%), Klebsiella spp. (5.7%), Candida spp. (4.1%), and Proteus spp. (1.5%). The isolated bacteria were mostly resistant to ampicillin (96.5%), sulphamethoxazole (96.5%), streptomycin (95.1%), oxytetracycline (85%), and amoxicillin (78.1%). However, the isolates were highly susceptible to enrofloxacin (86.2%), gentamicin (83.5%), and florfenicol (82.6%). This study might be helpful to the clinicians and researchers associated with the dairy industry for designing prophylactic as well as therapeutic strategies for the control of bovine mastitis. [ABSTRACT FROM AUTHOR]