Background: Tuberculosis (TB) control is more likely to be achieved if the level of knowledge regarding TB is increased among health workers managing high-risk groups. No formal assessments regarding knowledge, attitudes and practises of health workers about TB have been published for Mozambique, a country facing challenges in the fight against TB, with a fragile health system and considerable work overload of health personnel. The main objective of the study was to determine the level of knowledge, identify attitudes and assess practices regarding TB care and control among health care workers of the district of Manhiça. Methods: A descriptive cross-sectional study was performed through the use of a specifically designed Knowledge, Attitudes and Practices (KAP) questionnaire in the district of Manhiça, a high tuberculosis and HIV burden rural area in Southern Mozambique. In this district, 14 health care facilities service a population of approximately 160,000 people. The questionnaire took 30-45 min to administer with external assistance not permitted. The survey contained 79 questions pertaining to four different areas: demographics, TB knowledge, attitudes and practices. Results: The study sample included 170 health care workers. The average knowledge score was 14.89 points (SD = 3.61) out of a total possible 26 points. Less than 30% of respondents had heard of Xpert MTB/RIF®. Seventy per cent agreed there was stigma associated with TB and 48.2% believed this stigma was greater than that associated with HIV. The average practice score was 3.2 out of 9 points (35.6%, SD = 2.4). Conclusion: Health care worker's knowledge gaps identified in this study may result in substandard patient care. Specific deficiencies in understanding existed in terms of paediatric TB and Xpert MTB/RIF® testing. The present study provides impetus for tailored TB education among health care workers from a high TB burden rural area in Southern Mozambique.