Background The goal of antiretroviral therapy is to achieve sustained human immune deficiency virus (HIV) viral suppression. However, research on factors associated with viral load suppression among adolescents in low and middle-income countries is limited. The objectives of this study were to determine HIV viral suppression levels among adolescents in Kabale district and the associated clinical, adherence and psychosocial factors. Methods Cross-sectional and retrospective cohort study designs were used. Two hundred and forty-nine adolescents living with HIV that attended clinics between September and October 2019 at nine health facilities were interviewed and their medical records reviewed. A data abstraction tool was used to collect clinical data from adolescent’s clinical charts, face to face interviews were conducted using semi-structured questionnaire adopted from the HEADS tool and in-depth interviews conducted with ten key informants. Qualitative data was analyzed using thematic content analysis. Logistic regression was used to determine the magnitude by which clinical and psychosocial factors influence viral load suppression. Odds Ratios (ORs) were used for statistical associations at 95% confidence interval considering statistical significance for p-values less than 0.05. Qualitative data collected from Key informants to support our quantitative findings was analyzed using thematic content analysis. Results HIV viral suppression among (n = 249) adolescents was at 81%. Having no severe opportunistic infections was associated with viral load suppression among adolescent living with HIV (OR = 1.09; 95%CI [1.753–4.589]; p<0.001) as well as having no treatment interruptions (OR = 0.86; 95% CI [2.414–6.790]; p = 0.004). Belonging to a support group (OR = 1.01; 95% CI [1.53–4.88]; P = 0.020), having parents alive (OR = 2.04; 95% CI[1.02–4.12]; P = 0.047) and having meals in a day (OR = 5.68; C.I = 2.38–6.12, P = 0.010), were significantly associated to viral load suppression. The findings also indicated that long distances from health facilities, transport challenges and unprofessional conduct of health workers that make adolescent unwelcome at health facilities negatively affected viral suppression among adolescents. Conclusion The findings indicate that HIV viral suppression among adolescents on ART was at 81%. Kabale district was likely not to achieve the third 90 of the UNAIDS 90-90-90 global target for this population category. The findings further indicate that having no severe opportunistic infection and no treatment interruptions, good nutrition status, peer support and support from significant others, were highly associated with viral load suppression.