Glucose-6-phosphate dehydrogenase (G6PD), deficiency is considered the most common enzyme deficiency worldwide. Several studies have reported the frequency of G6PD deficiency in different regions of Saudi Arabia. This retrospective descriptive cross-sectional study was conducted at the International Medical Center Hospital in Jeddah, Saudi Arabia, which included families of children who had been diagnosed with G6PD deficiency between August 2016 and June 2021 at the pediatric hematology clinic. The data was collected by a developed questionnaire through the online platform. The study included 233 respondents. Results showed that most respondents responded that G6PD develops due to a genetic defect (98.7%). Knowledge scores did not differ significantly based on the child’s age and gender. However, mothers had a significantly higher median knowledge score than other relationship categories (p = 0.011). Additionally, younger age groups had significantly higher knowledge scores than older groups (p < 0.0001). Furthermore, the scores of relatives with a high school diploma and university degree were significantly lower than those with other educational levels (p = 0.046). The most reported perception of the causes and triggers of symptoms was eating legumes (91.0%). G6PD is a prevalent red blood cell disorder. Family and community awareness of the modes of inheritance, disease mechanisms, signs and symptoms, and management is critical to the overall well-being of children affected by this condition. Therefore, public education about this common condition is needed, especially in Middle Eastern communities.