Artemether-lumefantrine (AL) and artesunate-amodiaquine are first-line treatment for uncomplicated malaria in many endemic countries, including Mali. Dihydroartemisinin-piperaquine (DHA-PQ) is also an alternative first-line artemisinin-based combination therapy, but only few data are available on DHA-PQ efficacy in sub-Saharan Africa. The main aim of this study was to compare clinical efficacy of DHA-PQ versus AL, using the World Health Organization (WHO) 42-day in vivo protocol. The efficacy of three-dose regimens of DHA-PQ was compared to AL combination in a randomized, comparative open label trial using the WHO 42-day follow-up protocol from 2013 to 2015 in Doneguebougou and Torodo, Mali. The primary endpoint was to access the PCR-corrected Adequate Clinical and Parasitological Responses at day 28. A total of 317 uncomplicated malaria patients were enrolled, with 159 in DHA-PQ arm and 158 in AL arm. The parasite positivity rate decreased from 68.4% (95% CI 60.5-75.5) on day 1 to 3.8% (95% CI 1.4-8.1) on day 2 for DHA-PQ and 79.8% (95% CI 72.3-85.7) on day 1 to 9.5% (95% CI 5.4-15.2) on day 2 for AL, (p = 0.04). There was a significant difference in the uncorrected ACPR between DHA-PQ and AL, both at 28-day and 42-day follow-up with 97.4% (95% CI 93.5-99.3) in DHA-PQ vs 84.5% (95% CI 77.8-89.8) in AL (p < 0.001) and 94.2% (95% CI 89.3-97.3) in DHA-PQ vs 73.4% (95% CI 65.7-80.2) in AL, respectively (p < 0.001). After molecular correction, there was no significant difference in ACPRc between DHA-PQ and AL, both at the 28-day and 42-day follow-up with 99.4% (95% CI 96.5-100) in DHA-PQ versus 98.1% (95% CI 94.5-99.6) in AL (p = 0.3) and 99.3% (95% CI 96.5-100) in DHA-PQ vs 97.4% (95% CI 93.5-99.3) in AL (p = 0.2). There was no significant difference between DHA-PQ and AL in QTc prolongation 12.1% vs 7%, respectively (p = 0.4). The results showed that dihydroartemisinin-piperaquine and artemether-lumefantrine were clinically efficacious on Plasmodium falciparum parasites in Mali.