The prevalence of Type 2 diabetes (T2D) is increasing worldwide. Genetics and lifestyle, especially diet, are contributing factors. Analyses of macro- and micronutrient intake across global populations may help to explain their impact on glucose homeostasis and disease development. To this end, 420 Tunisians were enrolled in a prospective cross-sectional study of daily food consumption. Various data were collected and blood samples were drawn for biochemical assay. A 24-h recall questionnaire was obtained from participants to evaluate dietary intake. Statistical analyses were conducted using Nutrilog and R software. Biochemical analyses stratified the studied population (n = 371) into three groups: diabetics (n = 106), prediabetics (n = 192) and controls (n = 73); 49 subjects were excluded. Our results showed that Tunisians had hypercaloric diets high in carbohydrates and fat with variability in the levels of some vitamins and minerals, including riboflavin and niacin, that were statistically different among groups. The lower intake of vitamin D was associated with a greater risk of T2D. Higher vitamin A and sodium intake were associated with poor glucose homeostasis, although protein intake may improve it. In perspective, nutrigenomic studies can provide insight into problematic diets and poor eating habits and offer opportunities to analyze the effects of behavioral changes that can mitigate T2D development and progression.