Selenium is an essential element for the well-being of humans and animals. Micronutrient deficiencies affect over half of the global population. Biofortification of crops with selenium represents an economically sustainable solution to combat this problem. In this study, seven Allium species underwent Se biofortification, via Se soil enrichment with selenate in pots, at three distinct concentration levels and were used to explore the relationship between Se plant internalization, polyphenol composition, and mineral content. Allium species were chosen because they are recognized as secondary accumulators of Se. Plant morphology, as well as the presence of fifteen phenolic acids and flavonoids, were analysed using a UHPLC-DAD system. Mineral content was determined using both ICP-OES and FAES. Se levels were quantified using a validated method based on HG-HR-CS-QT-AAS. Even at the lowest biofortification level, Allium species demonstrated potential as sources of dietary Se, offering viable alternatives to Se supplements, without negatively impacting the main mineral content. Higher biofortification levels led to reduced levels of flavonoids and phenolic acids, accompanied by potential decreases in biomass for certain species. The study highlighted how Se concentration affects key polyphenols like quercetin, gallic acid, and luteolin, although the response varied among species.