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A healthcare worker's lack of knowledge of tuberculosis (TB) may contribute to their risk of developing the disease. With limited TB infection control activities in health institutions across Africa, there is a need to evaluate information challenges in TB services delivery from the front-line, in order to clarify priority areas for intervention. We examine the knowledge of TB among frontline healthcare workers in rural Nigeria. A cross-sectional study was conducted in December 2011 during an interactive clinical meeting organised for TB-focal persons in charge of 45 rural health facilities by the National Tuberculosis and Leprosy Control Programme in Ebonyi State. All the participants completed a self-administered questionnaire assessing knowledge on TB, treatment, and infection control. Participants with ⩾70% of correct responses were considered to have a good knowledge. Of the 52 healthcare workers surveyed, 48% were professionals (doctors/nurses) with a mean age of 36.3±10.6 years. Just over half of the respondents were female 27 (52%), most had tertiary education 43 (83%), and 33 (63%) had worked in TB care for 5 years or more. Only 14 (27%) of the respondents had a good knowledge score. Mean knowledge scores varied across job categories (f=6.2, p< 0.001) and with duration working in TB care (f =30.7, p< 0.001). Knowledge gaps existed regarding TB disease and treatment, but most deficits were found in infection control. TB knowledge gaps were found among rural healthcare workers, this may influence their risk of nosocomial TB infection: therefore they should be re-educated about TB, especially infection control. [ABSTRACT FROM AUTHOR]