Introduction. The morbidity and mortality from malaria are still unacceptably high in the developing countries, especially among the vulnerable groups like pregnant women and under-five children, despite all control efforts. The knowledge about the preventive measures of malaria is an important preceding factor for the acceptance and use of malaria preventive measures like Insecticide Treated Nets (ITN) by community members. Therefore, this study assessed the knowledge of malaria prevention among caregivers of under-five children and pregnant women in a rural community in Southwest Nigeria.Methodology. This is part of a larger malaria prevention study in rural Southwest Nigeria. A descriptive cross-sectional survey was conducted among pregnant women and caregivers of under-five children in Igbo-Ora, a rural town in Southwest Nigeria using a semi-structured, interviewer-administered questionnaire. Information was obtained on knowledge of malaria prevention, and overall composite scores were computed for knowledge of malaria prevention and ITN use. Data were analyzed using SPSS version 16. Associations between variables were tested using a Chi-square with the level of statistical significance set at 5%.Results. Of the 631 respondents, 84.9% were caregivers of under-five children and 67.7% were married. Mean age was 27.7 ± 6.3 years with 53.4% aged between 20 and 29 years. Majority (91.1%) had at least primary school education and 60.2% were traders. Overall, 57.7% had poor knowledge of malaria prevention. A good proportion (83.5%) were aware of the use of ITN for malaria prevention while 30.6% had poor knowledge of its use. Respondents who were younger (<30 years), had at least primary education and earn <10,000/per month had significantly poor knowledge of ITN use in malaria prevention. Majority (60.0%) respondents had poor attitude regarding use of ITNs.Conclusion. This study showed that the knowledge of malaria prevention is still low among under-five caregivers and pregnant women in rural Southwest Nigeria despite current control measures. There is a need for concerted health education intervention to improve the knowledge of rural dwellers regarding malaria prevention, including the use of ITN. This will go a long way to improving the reported low level of ownership and utilization of ITN in the rural areas.