Background: Ghana’s national tuberculosis (TB) prevalence survey in 2013 showed higher than expected prevalence, indicating people with TB are being missed. Aim: To identify barriers in the pathway to TB diagnosis and treatment initiation for people presenting to health facilities and make recommendations for improvement. Methods: In Volta region, Ghana, a cross-sectional study was conducted among symptomatic patients exiting the health facility to determine TB screening practices of healthcare workers (HCWs); a prospective cohort study among presumptive TB patients to determine if sputum was submitted; and in-depth interviews with HCWs and clinic observations to explore barriers to TB case finding. Results: In the cross-sectional study, 386/581 (66.4%) reported their TB-related symptoms to a HCW; 157/386 (40.6%) were eligible for a sputum test but only 31 (19.7%) were asked to submit a sputum. Prior TB treatment was the strongest predictor of being asked to submit a sputum (adjusted odds ratio [aOR]: 6.25, 95% CI: 2.24-17.46). In the prospective cohort study, among rural facility attendees, only 45/143 (31.5%) submitted a sputum for testing. Travel distance >10 km to the diagnostic laboratory was the strongest predictor of sputum not submitted (aOR 0.12, 95%CI 0.05-0.33). Barriers identified in the qualitative study were either health system- related such as no diagnostic laboratories in rural facilities and HCWs’ non-adherence to diagnostic guidelines; or HCW-related such as lack of training on guidelines, fear of infection leading to low motivation for TB work. Conclusions: There are gaps in the pre-diagnostic cascade in the steps before a sputum is submitted for a TB test. Factors causing these gaps are multifaceted and there is no single solution for these barriers. Collaborations between health facilities and the national TB control programme to implement patient-centred strategies for improved case finding are needed.