This study assessed the knowledge, attitudes, and practices (KAP) in malaria prevention using insecticide-treated bed nets (ITNs) among mothers of children under five years of age in White Nile State, Sudan. Multistage cluster sampling was used to select 761 mothers for this cross-sectional survey. There were gaps in the KAP in malaria prevention. Only 46.3% of the mothers stated personal protective measures (PPMs) as the best malaria prevention strategy; 54.9% considered ITNs as an effective means; and only 18.7% reported sleeping under an ITN every day. Older mothers were less likely to have good knowledge (OR=0.96), attitudes (OR=0.98) and practices (OR=0.98). Having a Government employee as the head of the household was positively associated with knowledge (OR=2.16) and attitudes (OR=1.96). The mother having a formal education was also positively associated with good knowledge (OR=1.55) and positive attitudes (OR=1.69). Mothers with a monthly household income of <491 Sudanese Pound were more likely to have good knowledge (OR=1.43). Mothers who had good knowledge (OR=4.99) and positive attitudes towards PPMs (OR=2.60) in malaria prevention were found to be more likely to practice preventive measures. Therefore, we recommend focusing on raising mothers’ awareness of the different preventive activities to protect them and their family from malaria.