Background: Malaria is a vector-borne infectious disease caused by eukaryotic protist of genus Plasmodium and transmitted by female Anopheles mosquitoes. Those living mainly in the world’s poorest countries are at risk of malaria as it is more endemic in the tropical and sub-tropical regions. Malaria spread have been linked to environmental changes, malaria vector dynamics, host immune status as well as individual or community factors such as the socio-economic status, knowledge of malaria, and the protective behavior. Objectives: To assess knowledge and practice of malaria prevention among non-medical students of higher institutions in Osun State, Nigeria. Materials and Methods: An open-ended structured questionnaire was administered consecutively to 1420 consenting non-medical students by the interviewer at various higher institutions. The cross-sectional survey questionnaire comprised sections on socio-demographic data, knowledge about causes of malaria, and knowledge about malaria prevention and practice. Results: The mean age (standard deviation) was 22.8 (12.7) years. 733 respondents (61.3%) are male while 462 respondents (38.7%) are female. 1124 students (94.1%) stated that malaria can be prevented. Out of those that stated that malaria can be prevented listed methods to prevent malaria to include reducing exposure to mosquitoes by use of insecticide treated mosquito nets and indoor residual spraying. 570 students (47.7%) reported that malaria is more common in the tropical region. Only 181 (15.1%) students know the cause of malaria in humans to be Plasmodium. 1013 (84.3%) reported malaria to breed more during the rainy season and 473 (39.6%) students reported female Anopheles mosquito to be responsible for malaria. Conclusion: Non-medical students have relatively reasonable knowledge about malaria preventive measures and practices, although, certain aspects are still not well-understood, probably due to the scope of their educational curricula. Hence, there is a need to intensify malaria public enlightenment programs and promote affordable preventive measures.