Bovine Mastitis is a responsible for the most economic lost to dairy producers and the milk industry, because it decreases the production of milk and deteriorates the quality. The objective of the present study was to determine the prevalence on different etiological infectious agents, which are fault of causing mastitis on milky specialized farms in the Boyacense Highlands-Colombia. In this project, the researchers took into account 30 milky livestock farms, located between 2300 and 2800 meters over the sea level. Every farm uses a mechanical milking and had two milky gatherings by day. One hundred percent of the cattle were Holstein Friesian and their main food source was kikuyu Grass (Pennisetum clandestinum). This study took a time of three years. In that term, a total of 5,396 quarters were evaluated in 1,349 milking cows through California Mastitis Test (CMT). Subclinical mastitis was presented in 3.25% of lactating cows. The main bacteria isolated with bacteriological routine examinations, over the three years of the study, were: Streptococcus agalactiae (9.7%); others Streptococcus (4.1%); Staphylococcus aureus (8.3%); others such as: Staphylococcus positive coagulase (0.55%); Staphylococcus negative coagulase (0.46%); Actinomyces pyogenes (1.3%); yeast (0.7%); Escherichia coli (0.6%); Corynebacterium (0.6%); mixed additives Strep.+ Staphy. (0.8%) Other mixed additives (4.5%) Acholeplasma (1.4%) and Mycoplasmas spp (2.2%). The last two were reported for the first time in Colombia.