Accurate diagnosis of malaria is important for providing malaria burden and preventing misdiagnosis. To analyses the diagnostic performance of conventional blood film microscopic examination, OptiMAL Rapid Antigen Test, Hexagon Malaria Combi, Diaquick Malaria Pf/Pan Cassette in comparison with species-specific nested polymerase chain reaction (nest-
PCR) in Makkah and Taif in Kingdom of Saudi Arabia. A total of 100 blood samples collected from febrile cases in Makkah and Taif Cities were screened for malaria by microscopic examination, rapid diagnostic tests (RDTs) using nest-PCR as standard method. The results showed differences in detection rates of nested-PCR, conventional blood film microscopic examination, OptiMAL test, Hexagon Malaria Combi, Diaquick Malaria Pf/Pan Cassette (72%, 57%, 59%, 44% & 52% respectively) in 100 clinically suspected cases. Microscopic examination, OptiMAL test, Hexagon Malaria Combi and Diaquick Malaria Pf/Pan Cassette showed sensitivity (72.2%,72.2%, 62.9%, 65.3%), specificity (82.1%, 75%, 82.1%,& 82.1%), accuracy index (75%, 73%, 62%, 70%), positive predictive value (91.2%, 88.1%, 88.6%,& 90.4%) and negative predictive value (53.5%, 51.2%, 41.1%, 47.9) using nested-PCR as the reference technique. The detection rates of nested-PCR for Pasmodium infection was superior than conventional blood film microscopic examination, OptiMAL test, Hexagon Malaria Combi microscopy and Diaquick Malaria Pf/Pan Cassette. The nest-PCR method was more reliable than conventional microscopic examination for the diagnosis of malaria infections, and this is particularly true in cases of mixed infections and submicroscopic infections.