Tuberculosis remains a leading cause of death worldwide. Transmission is the dominant mechanism sustaining the multidrug-resistant tuberculosis epidemic. Tuberculosis infection prevention and control (TBIPC) guidelines for healthcare facilities are poorly implemented. This systematic review aimed to explore the barriers and facilitators of implementation of TBIPC guidelines in low- and middle-income countries from the perspective of healthcare workers. Two separate reviewers carried out an electronic database search to select qualitative and quantitative studies exploring healthcare workers attitudes towards TBIPC. Eligible studies underwent thematic synthesis. Derived themes were further organised into a macro-, meso- and micro-level framework, which allows us to analyse barriers at different levels of the healthcare system. We found that most studies focused on assessing implementation within facilities in accordance with the hierarchy of TBIPC measures-administrative, environmental and respiratory protection controls. TBIPC implementation was over-estimated by self-report compared with what researchers observed within facilities, indicating a knowledge-action gap. Macro-level barriers included the lack of coordination of integrated HIV/tuberculosis care, in the context of an expanding antiretroviral therapy programme and hence increasing opportunity for nosocomial acquisition of tuberculosis; a lack of funding; and ineffective occupational health policies, such as poor systems for screening for tuberculosis amongst healthcare workers. Meso-level barriers included little staff training to implement programmes, and managers not understanding policy sufficiently to translate it into an IPC programme. Most studies reported micro-level barriers including the impact of stigma, work culture, lack of perception of risk, poor supply and use of respirators and difficulty sensitising patients to the need for IPC. Existing literature on healthcare workers' attitudes to TBIPC focusses on collecting data about poor implementation at facility level. In order to bridge the knowledge-action gap, we need to understand how best to implement policy, taking account of the context.