Abstract Background Decay of HIV in seminal plasma (SP) and rectal fluid (RF) has not yet been described for the antiretroviral combination of dolutegravir (DTG) + lamivudine (3TC). Methods In this randomized multicenter pilot trial, males who were antiretroviral naive were randomized (2:1) to DTG + 3TC or bictegravir/emtricitabine/tenofovir alafenamide (BIC/FTC/TAF). HIV-1 RNA was measured in blood plasma (BP), SP, and RF at baseline; days 3, 7, 14, and 28; and weeks 12 and 24. Results Of 25 individuals enrolled, 24 completed the study (DTG + 3TC, n = 16; BIC/FTC/TAF, n = 8). No significant differences were observed between groups for median decline in HIV-1 RNA from baseline at each time point or median time to achieve HIV-1 RNA <20 copies/mL in BP and SP and <20 copies/swab in RF. HIV-1 RNA decay patterns were compared in individuals receiving DTG + 3TC. Despite significantly higher percentages for changes from baseline in BP, median (IQR) times to HIV-1 RNA suppression were shorter in SP (7 days; 0–8.75) and RF (10.5 days; 3–17.5) than in BP (28 days; 14–84; P < .001). Conclusions Comparable HIV-1 RNA decay in BP, SP, and RF was observed between DTG + 3TC and BIC/FTC/TAF. As shown with triple-drug integrase inhibitor–based regimens, rapid HIV-1 RNA suppression in SP and RF is achieved with DTG + 3TC, despite decay patterns differing from those of BP. Clinical Trials Registration EudraCT 2019-004109-28.