Background: Spina bifida is one of the most challenging and partially preventable congenital diseases that can be prevented with adequate amounts of folic acid (FA) intake before conception and throughout the first few months of pregnancy. Knowledge about the adequate timing and duration of FA intake is critical for prevention. Methods: A questionnaire-based, cross-sectional study was conducted to assess spina bifida and FA awareness among female students at King Khalid University (KKU), Abha, Saudi Arabia. The study included three groups: health-college female students (HCS), non-health-college female students (NHCS), and KKU female employees (others). These three groups were attendants at the Spina Bifida Awareness Week that was held at KKU in March 2017. Results: There were 1,366 participants, of whom 11% were HCS, 59% were NHCS, and 30% were "others". Across all tested domains (i.e., spina bifida awareness, FA intake time and duration, diseases prevented by folic acid intake, and food rich in folic acid), it was found that statistically significant differences existed. The results showed a highly significant lack of knowledge towards the aforementioned domains in both the NHCS and "others" groups. Marital status and number of children were independent variables and showed statistically significant differences in knowledge toward the tested domains. Conclusion: In Saudi Arabia, more attention should be paid to educate women of childbearing age on the importance of FA intake in preventing spina bifida. These results indicate how FA intake awareness should be embedded in junior-high and secondary school curricula to further educate females on the importance of FA.