Pearl millet is a climate-resilient crop suitable for arid regions due to its ability to tolerate drought and severe climatic conditions. It is a nutritious grain with potential health-promoting properties that can contribute to the world food security and livelihoods for small-scale farmers. In Tunisia, this crop has historically possessed a diverse and rich germplasm, but it now faces the threat of genetic erosion. The preservation programs for these species have been infrequent and have not attributed the plant its due value, resulting in a period of neglect and lack of proper monitoring. To rejuvenate this field, efforts are underway to revitalize research and development initiatives, as part of these efforts, seeds collected from the southern Tunisian regions of Medenine and Djerba were sown in the experimental plot of the Arid Zones Institute for 2 years. Subsequently, the harvested grains were processed and subjected to biochemical analyses. The aim of this study was to assess the profiles of phenolic compounds, sugar content, fatty acids, and mineral composition in ten pearl millet genotypes. An LC-MS analysis allowed for the identification of various phenolic acids in millet grains, with quinic acid, p-coumaric acid, and caffeic acid emerging as the predominant phenolic acids. Furthermore, two flavonoid compounds were identified, quercetin and apigenin. The analysis of sugar contents by LC-MS showed an abundance of glucose and fructose, followed by maltose and sucrose. Mean values ranged between 323.98 mg/kg for G4 and 2163.86 mg/kg for G8. The minerals K, Mg, Na, and Ca were detected in interestedly high quantities. GC-MS analysis showed the detection of seven fatty acids. Arachidic acid (0.53-2.23%) and linolenic acid (1.01-2.23%) were present in relatively abundant amounts. Linoleic acid and linoleic acid were the most frequently occurring fatty acids. When examining the variability among the assessed attributes, heatmap analysis revealed correlations between each trait and the clustered genotypes. Our findings indicate that pearl millet serves as a rich source of diverse bioactive molecules, making it an interesting material for potential industrial applications.