Background: The enzootic bovine leukosis (EBL) is a viral disease with a chronic aspect and its etiological agent is an oncogenic deltaretrovirus called bovine leukemia virus (VLB). It has a wide distribution in dairy cattle herds, and it may take several years before the first clinical manifestations occur. Animals seropositive for VLB are more susceptible to developing infectious diseases such as mastitis. The objective of this study was to evaluate the seroprevalence of EBL, the prevalence of mastitis, the possible association between leukosis and mastitis, and the risk factors related to leukosis in municipalities of the Caparaó region of Espírito Santo, Brazil.Materials, Methods & Results: The prevalence of enzootic bovine leukosis and its association with mastitis were evaluated in an 854 clinically healthy crossbred (Bos tauros tauros x Bos taurus indicus) dairy cows were used, in different lactation phases, from farms located in the 12 municipalities that compose the Caparaó Capixaba micro-region, in the southern part of Espírito Santo, Brazil, in the period from February to July 2015. The seroprevalence of EBL was determined by the agar gel immunodiffusion test, the presence of clinical mastitis was ascertained by the black background mug test and of subclinical mastitis by the CMT. The reuse of needles, type of milking, reproductive management, calf mortality, presence of reproductive problems and milk yield were the risk factors assessed. The association between the variables was estimated by the odds ratio (95% confidence interval). The significance of the associations was determined by the Chi-square test. The prevalence of EBL, clinical and subclinical mastitis was 56.79%, 5.50% and 43.55%, respectively. There was a positive association between the EBL virus and the presence of subclinical mastitis (P < 0.05, OR = 1.41, CI = 1.07-1.86). The reuse of disposable needles (OR = 1.92, CI = 1.46-2.53), mechanical milking (OR = 2.03, CI = 1.34-3.08), calf mortality (OR = 2.39; CI = 1.25-4.55) and the presence of reproductive problems (OR = 1.62; CI = 1.23-2.14) were considered risk factors associated with LEB (P < 0.05).Discussion: Enzootic bovine leukosis is disseminated in the dairy herds in the municipalities the study. The lack of knowledge about EBL among farmers and the failure to adopt measures for its control and elimination favor the appearance of new cases in the herd. Factors such as animal age, absence of exams when purchasing animals, reuse of gloves for rectal palpation and needles, contribute to dissemination of the infection. The economic losses associated with subclinical mastitis can even be greater than those caused by clinical mastitis. The differences for prevalence of clinical mastitis can be associated with the breeding system, type of management, intensification and level of production. In this context, we noted a much larger number of animals with subclinical infection, where the losses may not be perceptible to the farmers. It is concluded that EBL is widespread in dairy herds of the municipalities of Caparaó Espírito Santo's region. The prevalence of clinical and subclinical mastitis is high in some cities surveyed. The presence of EBL increases the chances of developing subclinical mastitis. The reuse of disposable needles and mechanical milking were considered risk factors associated with EBL. Seroreagent animals for EBL are more likely to present calf mortality and reproductive problems. It is necessary to discuss the best sanitary management system for the region, with a better economic and social response and then carry out the elaboration of educational and implementation measures, through technical assistance.