Mastitis is one of the most important diseases for the dairy industry worldwide due to the great economic losses it causes, and the bacterial agents that it causes variation from one herd to another. The objective of the investigation was determined the infectious agents involved in the development of mastitis in specialized dairies herds in the Highlands of Boyacá, Colombia. Was performed CMT tests in 4432 teats to determine the positive results for inflammation (CMT + 2), sampling in aseptic material of positives quarters, identification and differentiation of through microbiological cultures, Gram staining, and biochemical tests of bacterial and fungal agents presents in milk samples. It was determined that prevalence´s varied between the years of the study, in 2016 there was a total prevalence of 45.19%, in 2017: 51.06% and in 2018: 39.76%. The months of April, May, and September, October presented the highest number of teats affected with mastitis during the three years of study. Streptococcus agalactiae and Staphylococcus aureus were the most prevalent microorganisms that cause mastitis in the three study years. There was an increase in the prevalence of infected teats corresponding to the months (April, May, September, and October) due to rainfall increases. The contagious pathogens were most prevalent that environmental pathogens.