Bovine mastitis is the most common and major economic problem in the dairy industry worldwide, with a wide variety of microorganisms involved. Identification of mastitis pathogens is important for the selection of appropriate antimicrobial therapies. Antimicrobial agents are used to treat these infections caused by bacteria in particular but in recent years some bacteria have demonstrated full or partial resistance to different antibiotics. This phenomenon called antimicrobial resistance is a rising concern in both public and animal health and in this case, dairy science. The objective of the research was to determine the prevalence and antimicrobial resistance of pathogens involved in bovine mastitis in a region of Colombia. 1392 quarters of 348 cows from Anaime Colombia region were tested, using the California mastitis test (CMT). The positive samples to CMT were cultured for bacteriological isolation and were tested for antibiotic susceptibility and resistance by disk diffusion method and performed according to CLSI guidelines in Mueller-Hinton agar. Analysis of the results was made through descriptive statistics and prevalence ratio. 190 (54,6%) cows were considered mastitis free. 158 cows (45,4%) were positives for CMT and bacteriological culture. Clinical mastitis in 20 (5,74%) cows characterized by milk abnormalities and subclinical mastitis in 138 (39,65%) were found. Coagulase negative staphylococci (CoNS) was the main pathogenic agent with 44,79% of the total isolations, followed by Staphylococcus aureus (CoNP) 30.73% and Streptococcus spp. 22.39%, both Klebsiella pneumoniae and E. coli with 1.1%. CoNS and S. aureus exhibited the highest degree of resistance to penicillin G, S. aureus and Streptococcus spp. showed a high resistance to streptomycin and erythromycin respectively. The most prevalent organism was CoNS considered currently a worldwide emerging mastitis and it was mainly evidenced as a cause of subclinical mastitis with high degree of resistance to penicillin.