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Tuberculosis is a global public health problem. Lesotho, a sub-Saharan country with high HIV and tuberculosis burden, launched the Three I’s programme of the World Health Organization in 2013. However, the outcomes of this intervention are scarcely known. This study evaluated the profile of tuberculosis diagnostic techniques used, treatment outcomes and the associated factors across the baseline period (2010–2012) and after the launch of the Three I’s programme (2013–2015) based on a retrospective cohort review of patient records at a major clinic in Maseru, Lesotho. In total, 812 cases with complete records from 1 066 randomly selected cases treated (every second case from the sampling frame) were included in the study. Factor analysis was based on logistic regression analysis. The use of the GeneXpert MTB/RIF technique remained below 20% throughout the study period. Overall, 28.9% (n = 812) cases had unsuccessful tuberculosis treatment. Male gender (p = 0.046), extra-pulmonary tuberculosis (p = 0.002) and treatment observation by community health workers (p = 0.001) were significantly associated with unsuccessful treatment outcome. Overall, treatment outcomes did not differ significantly (p = 0.636) before and after the launch of the intervention. These findings indicate the need to strengthen the implementation of the Three I’s programme, taking into account the significant factors in this study.