Background: Information on the prevalence of bovine brucellosis and tuberculosis in West African countries other than Nigeria had been scanty in literature. This study estimates the prevalence of brucellosis and bovine tuberculosis in cattle from selected herds in Dormaa and Kintampo Districts, Brong Ahafo region, Ghana. Methods and Findings: The study involved testing of 444 cattle for tuberculosis and brucellosis using single comparative intradermal tuberculin and Rose Bengal plate tests respectively. Tuberculosis was recorded in 29 cattle giving overall individual cattle level tuberculosis prevalence of 6.5% and herd-level prevalence was 64.7Â±4.0% while the positive reaction to Rose Bengal was recorded in 13 cattle with overall individual cattle level brucellosis prevalence of 2.93%, the herd prevalence was 35.3Â±3.0%. The positive reactors to the diseases were significantly more in Dormaa district and cows than bulls. The infection was detected with increasing age, more between age 1 to 4 cattle in both diseases. The two diseases were detected in a >5year old cow giving an estimated prevalence of 0.23%. Conclusions: Though the estimated prevalence of the two diseases was relatively low in the districts investigated, surveillance and prevention of the disease in Ghana may be warranted because of the continual unrestricted animal movement which might result in an increased transmission to humans.