Background TB is ranked second among the leading chronic illnesses in causing morbidity and mortality after HIV the human immunodeficiency virus (HIV). Despite the relative success in the implementation of the DOTS in Uganda, challenges still persist that have hindered the required standards. Rubaga division has registered a significant reduction in the proportion of patients completing treatment from 47% to 31% but this marked with increase in the defaulter rate of 4%. This discrepancy raises questions about the implementation of TB DOT in this division that need immediate answers. Objective The main objective of the study was to determine the factors affecting the implementation of TB DOT in Rubaga division as to improve patients’ adherence to TB treatment. Results A total of 201 patients and three TB clinic in-charges participated in the study. Out of the 201 patients, 66% reported their treatment was being observed by someone. The most commonly (82%) used mode of DOT is relative/family member not community-based treatment supporters. More than half, (53%) of the respondents had taken medication without treatment observers. Staying alone (have no relative/family member) in the house was the main reason why patients were not on TB DOT. Patient with positive attitude towards TB DOT and those in union were more likely to be on TB DOT. Follow-up of treatment observers was poor. Lack of treatment supporters down in the communities and poor facilitation of health workers were the main barriers affecting their practices during the implementation of TB DOT Conclusion Implementation of TB DOT is being done in Rubaga, relatives/ family members are the common treatment observers. Lack of community-based treatment supporters and poor facilitation of health worker are the leading barriers to the implementation of TB DOT in Rubaga division. Recommendation There is need for vigorous introduction of community-based treatment supporters to supplement the family members/relatives.

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