Background: Transfusion-transmitted malaria (TTM) remains a significant public health concern in malaria-endemic regions such as Ghana. The persistence of Plasmodium falciparum in donated blood, often undetected, poses a risk to transfusion recipients. This study evaluates the diagnostic accuracy of Rapid Diagnostic Tests (RDTs) and microscopy in detecting P. falciparum in donor blood. Aim: To compare the diagnostic performance of RDTs and microscopy in detecting P. falciparum in stored blood samples at SDA Hospital, Accra. Methods: A cross-sectional study was conducted on 115 blood samples donated to SDA Hospital. The samples were screened using First Response RDT kits and Giemsa-stained microscopy (gold standard). Sensitivity, specificity, and parasite density were analyzed using SPSS version 26. Results: Of the 115 samples, microscopy detected P. falciparum in 8.7% of the cases, while RDTs detected 6.1%. Microscopy showed 100% sensitivity and specificity, while RDTs demonstrated 100% sensitivity and 99.1% specificity. Blood group O positive was most frequently associated with parasitemia. All infected donors were male, with parasite densities ranging from 600 to 1200 parasites/µL. Conclusion: Both RDTs and microscopy showed high diagnostic performance, but microscopy detected more positive cases. Given the residual risk of TTM, integrating both diagnostic methods in screening protocols is recommended to improve transfusion safety in malariaendemic regions.