We studied the prevalence of bovine tuberculosis (BTB) in Mejia canton, the major dairy cattle production area in northern Ecuador. Twenty dairy herds comprising 2,022 cattle were selected. In 2007, each animal was tested using the comparative intradermal tuberculin test (CITT). In 2008, a follow-up test was performed in the same herds. The true annual incidence was 1.70%, and the true prevalence was 7.41% and 7.13% in 2007 and 2008, respectively. The prevalence was 0.27% and 0.57% in medium-sized herds in 2007 and 2008, respectively, compared with 8.63% and 8.43% in large herds (P < 0.01). The number of skin test-positive cases also increased significantly with age (P = 0.03), contacts with other species of animals (P < 0.01), and introduction of new cattle (P = 0.04). Herd prevalence was 55% in 2007 and 65% in 2008. This study shows the lack of knowledge in cattle farmers about this zoonosis and the necessity for a national BTB control program in Ecuador.