Background The World Health Organization recommends dolutegravir-based antiretroviral therapy (ART) as the preferred first-line regimen for HIV treatment. This retrospective cohort study evaluated the long-term virologic outcomes and safety of transitioning from an efavirenz-based regimen (tenofovir, lamivudine, efavirenz [TLE]) to a dolutegravir-based regimen (tenofovir, lamivudine, dolutegravir [TLD]) among adult HIV participants in Mbeya, Tanzania. Methods Medical records of 250 adult HIV participants who transitioned from TLE to TLD at Mbeya Zonal Referral Hospital were reviewed from August 2022 to December 2022. The primary outcome was virologic failure, defined as HIV RNA > 1000 copies/mL. Secondary outcomes included viral suppression (< 50 copies/mL) and adverse drug reactions (ADRs). Using appropriate statistical tests, participant characteristics and outcomes were compared before and six months after transitioning. Results At baseline on TLE, 88% had viral suppression, and 3.6% had virologic failure. Six months after transitioning to TLD, viral suppression was 87.2% and virologic failure increased to 6.8%. Overall, 79.6% experienced ADRs with TLD, predominantly neurological effects and weight gain. No significant associations were found between viral load changes and participant characteristics like age, sex or treatment duration. Conclusions Transitioning to dolutegravir maintained high rates of viral suppression comparable to efavirenz, albeit with a slight increase in virologic failure. Dolutegravir was well-tolerated overall despite a high ADR rate. Findings support the ongoing scale-up of dolutegravir in Tanzania and other resource-limited settings while highlighting the need for continued viral load monitoring and pharmacovigilance.