Malaria significantly impacts global populations, especially in sub-Saharan Africa, where Plasmodium falciparum predominates. The spread of drug-resistant parasites complicates treatment, necessitating effective disease management and control strategies. The study aims to evaluate malaria infection and its prevalence among symptomatic patients in Ota, Ogun State, Southwestern Nigeria healthcare centers. It employs a cross-sectional study design and random sampling to collect data. The investigation was conducted from September 2023 to February 2024. A total of three hundred (300) febrile subjects were examined. Under the aseptic condition, blood samples were obtained from each febrile patient to detect the presence of malaria parasites following standard parasitological methods. This involves examining thick and thin blood films stained with Giemsa and viewing them under the microscope. Out of 300 subjects examined, 136 (45.3%) were males, while 164 (54.6%) were females. 132 (44.0%) patients tested positive for malaria infection and 168 (56%) patients tested negative for malaria infection; only Plasmodium falciparum was detected as the species of Plasmodium present in the study area. There were 132 positive individuals with 71 male and 61 female subjects. The observed prevalence of malaria infection was 44.0%, although it differed based on gender and age. Males had significantly higher malaria prevalence rates (52.2%) than females (37.2%), higher among older children of 11–20 years (68.2%) and young adults of 21–30 years (59.5%), with the lowest prevalence rates recorded among the elderly above 60 years (20.8%). The malaria prevalence rate observed in this study indicates a significant difference (P ≤ 0.05) and an association with patient gender and age categories. The study established a comparatively low prevalence rate compared to other studies from this region, attributable to the development of significant levels of acquired immunity among residents stemming from frequent exposure to mosquito vectors. It, however, revealed higher cases among older children, young adults, and males of malaria infection within the study location, emphasizing the urgency for effective healthcare planning and intervention strategies to combat this escalating challenge, especially among these susceptible individuals with prioritized vector control programs and integrated healthcare services.