Introduction <p>Faba bean (<i>Vicia faba</i> L.) leaves are edible; hence, they are primarily used as animal feed in agriculture. Additionally, seed pods and other plant tissues are considered edible and are used as green vegetables in many parts of the world.</p> Objectives <p>Flavonol glycosides are well-known in faba bean leaves; accordingly, in this study, we followed a targeted metabolomic approach to explore glycosylated flavonols and their concentrations in response to contrasting levels of selenium (Se) and sulfur (S) enrichment under faba bean–<i>Rhizobium </i>symbiosis.</p> Methods <p>Faba bean plants were cultivated under growth chamber conditions and enriched with different levels of selenium and sulfur under <i>Rhizobium</i> inoculation. Their leaves were extracted using 70% methanol to quantify glycosylated flavonoids. Sample leaves were analyzed through a targeted method using high-performance liquid chromatography combined with a diode array detector (HPLC-DAD) and electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry detection (HPLC-ESI-Q-ToF-MS/MS).</p> Results <p>The analysis led to semi-quantifying 11 flavonol glycosides. Analysis of the metabolites of the different faba bean leaf extracts confirmed that selenium has a considerable impact on the accumulation of flavonol glycosides, especially under sulfur availability, possibly because it induces chalcone synthase and other enzymes for flavonols’ biosynthesis.</p> Conclusion <p>To the best of our knowledge, this is the first report to investigate the impact of selenium and sulfur enrichment on the accumulation of faba bean flavonols under atmospheric nitrogen (N₂) fixation conditions. This study highlights the medicinal and nutritional benefits of legumes as an essential source of protein in plant-based foods.</p>