A cross-sectional survey was conducted in selected districts of Bangladesh to estimate the prevalence of bovine tuberculosis (bTB), and to identify the risk factors for bTB. We included 1865 farmed cattle from 79 herds randomly selected from five districts. Herd and animal level data were collected using semi-structured interviews with cattle herd owners. The single intradermal comparative tuberculin test (SICTT) was used to estimate the prevalence of bTB. The risk factors were identified using mixed-effect multiple logistic regression analyses. The overall herd and animal level prevalences of bTB were estimated to be 45.6% (95% Confidence Interval [CI] = 34.3-57.2%) and 11.3 (95% CI = 9.9-12.8%), respectively, using the OIE recommended >4 mm cut-off. The true animal level prevalence of bTB was estimated to be 11.8 (95% Credible Interval = 2.1-20.3%). At the herd level, farm size, bTB history of the farm and type of husbandry were significantly associated with bTB status in univariable analysis. Similarly, age group, sex, pregnancy status and parity were significantly associated with bTB at cattle level. However, in multivariable analysis only herd size at the herd level and age group and pregnancy status at the cattle level were significant. Compared to a herd size of 1-10, the odds of bTB were 22.8 (95% CI: 5.2-100.9) and 45.6 times (95% CI: 5.0-417.7) greater in herd sizes of >20-50 and >50, respectively. The odds of bTB were 2.2 (95% CI: 1.0-4.5) and 2.5 times (95% CI: 1.1-5.4) higher in cattle aged >3-6 years and > 6 years, compared to cattle aged ≤1 year. Pregnancy increased the odds of bTB by 1.7 times (95% CI: 1.2-2.4) compared to non-pregnant cattle. Taken together, the results suggest high herd and animal level prevalence of bTB in these 5 districts, with the greatest risk of bTB in older and pregnant cattle within large herds (>20), and highlight an urgent need for continued surveillance and implementation of bTB control programs in Bangladesh.