ABSTRACT Malaria remains a leading cause of morbidity and mortality in Burkina Faso, which utilizes artemether–lumefantrine as the principal therapy to treat uncomplicated malaria and seasonal malaria chemoprevention with monthly sulfadoxine–pyrimethamine plus amodiaquine in children during the transmission season. Monitoring the activities of available antimalarial drugs is a high priority. We assessed the ex vivo susceptibility of Plasmodium falciparum to 11 drugs in isolates from patients presenting with uncomplicated malaria in Bobo-Dioulasso in 2021 and 2022. IC ₅₀ values were derived using a standard 72 h growth inhibition assay. Parasite DNA was sequenced to characterize known drug resistance-mediating polymorphisms. Isolates were generally susceptible, with IC ₅₀ values in the low-nM range, to chloroquine (median IC ₅₀ 10 nM, IQR 7.9–24), monodesethylamodiaquine (22, 14–46) piperaquine (6.1, 3.6–9.2), pyronaridine (3.0, 1.3–5.5), quinine (50, 30–75), mefloquine (7.1, 3.7–10), lumefantrine (7.1, 4.5–12), dihydroartemisinin (3.7, 2.2–5.5), and atovaquone (0.2, 0.1–0.3) and mostly resistant to cycloguanil (850, 543–1,290) and pyrimethamine (33,200, 18,400–54,200), although a small number of outliers were seen. Considering genetic markers of resistance to aminoquinolines, most samples had wild-type PfCRT K76T (87%) and PfMDR1 N86Y (95%) sequences. For markers of resistance to antifolates, established PfDHFR and PfDHPS mutations were highly prevalent, the PfDHPS A613S mutation was seen in 19% of samples, and key markers of high-level resistance (PfDHFR I164L; PfDHPS K540E) were absent or rare (A581G). Mutations in the PfK13 propeller domain known to mediate artemisinin partial resistance were not detected. Overall, our results suggest excellent susceptibilities to drugs now used to treat malaria and moderate, but stable, resistance to antifolates used to prevent malaria.