Light microscopy and rapid diagnostic tests are the two commonly used methods for malaria diagnosis that rely on the direct use of unprocessed blood samples. However, both methods do not have the level of sensitivity required for malaria diagnosis in cases of low density parasitaemia. We report here the diagnostic performance of a whole blood-based reverse transcription loop-mediated isothermal amplification method for Plasmodium falciparum malaria diagnosis in apparently healthy blood donors and febrile neonates in Cameroon. The presence of malaria parasites in whole blood samples was determined by light microscopy, antigen-based rapid diagnostic test (RDT), and by RT-LAMP using a "lyse and amplify" experimental protocol. Of the 256 blood donors tested, 36 (14.1%) were positive for malaria parasites by light microscopy, 38 (14.8%) were positive by RDT whereas 78 (30.5%) were positive by RT-LAMP. Only light microscopy and RT-LAMP detected infection among the febrile neonates (279 neonates, median age: 2 days, range: 1-9 days), with positivity rates of 8.6% and 12.2%, respectively. The overall concordance between the three methods were 75.9% for RT-LAMP and light microscopy, 75.1% for RT-LAMP and RDT, and 83.9% for light microscopy and RDT. Blood parasite densities were significantly lower in the neonates (mean: 97.6, range: 61-192 parasites/μL) compared to the blood donors (mean: 447.8, range: 63-11 000 parasites/μL). Together, the study demonstrates the usefulness of whole blood RT-LAMP for use in rapid pre-screening of blood donors and suspected neonates to avert severe consequences of P. falciparum infections.