Malaria remains a significant public health concern in Madagascar. The WHO recommends using parasitological methods to confirm Plasmodium infection before treatment. This study evaluated the performance of two rapid diagnostic tests (RDTs), Bioline™ Malaria Ag Pf/Pan (Abbott Point of Care, Princeton, NJ) and Bioline™ Malaria Ag Pf/Pv (Abbott Point of Care, Princeton, NJ), compared with microscopy and polymerase chain reaction (PCR) as reference methods. A prospective, diagnostic performance study was conducted in two malaria-endemic districts with different epidemiological contexts: Ifanadiana (Plasmodium falciparum [P. falciparum] transmission) and Mandoto (Plasmodium vivax transmission). Symptomatic patients and asymptomatic individuals aged 3-81 years were enrolled between April and July 2023. Finger prick blood samples were used for RDTs, Giemsa-stained blood films, and molecular analysis. Among 675 participants (401 symptomatic, 274 asymptomatic), the performance for detecting P. falciparum and non-falciparum/vivax malaria in symptomatic patients for Bioline™ Malaria Ag Pf/Pan was 96.5% (95% CI: 93.4-98.4%) and 91.5% (95% CI: 82.5-96.8%) sensitivity and 76.0% (95% CI: 64.7-85.1%) specificity, and the performance for Bioline™ Malaria Ag Pf/Pv was 95.0% and 91.5% sensitivity and 76.0% specificity. In asymptomatic individuals, the performance was 90.3% (95% CI: 83.4-95.0%) and 33.3% (95% CI: 4.3-77.7%) sensitivity and 85.4% (95% CI: 78.9-90.6%) specificity for Bioline™ Malaria Ag Pf/Pan and 80.6% (95% CI: 80.1-93.1%) and 0% (95% CI: 0-84.2%) sensitivity and 86.0% (95% CI: 79.7-91.0%) and 86.1% (95% CI: 79.7-91.0%) specificity for Bioline™ Malaria Ag Pf/Pv. Rapid diagnostic test performance varies with local epidemiology in symptomatic patients. The results emphasize the need for careful consideration of RDT use based on local epidemiology and clinical context. Both RDTs could be used when microscopy and PCR are unavailable.