A cross-sectional study was undertaken to determine the prevalence of Mycobacterium bovis skin positivity and associated risk factors in cattle in western Uganda. Herds were selected using multi-stage cluster sampling. The comparative cervical intradermal tuberculin test (CCT) was used to determine cattle tuberculosis status using US Department of Agriculture protocols. Risk factor data were collected from cattle owners through questionnaires collected by in-person interviews. Multivariable logistic regression models were used to measure the association between risk factors and herd CCT reactor prevalence. A total of 525 cattle from 63 herds were screened for M. bovis infection. Of the 525 cattle tested, 2.1 % were CCT reactors and 15.43 % were CCT suspects. Of herds tested, 14.28 % had at least 1 CCT reactor. Using a private water source for cattle and not introducing new cattle into the farm were associated with lower prevalence of M. bovis skin positivity. The herd-level prevalence of M. bovis reactors in Kashaari County of Mbarara District was 14.5 %, and the individual cattle prevalence was low (2.1 %). Using communal sources of drinking water for cattle and introducing new cattle on the farm were farm management practices associated with increased risk of M. bovis exposure in cattle. Despite the low prevalence of bovine tuberculosis (TB), there is a need to educate the populace on the possibility of human infection with zoonotic TB and for educating farmers on practices to reduce the risk of acquiring M. bovis in the Mbarara District.