AbstractMany bioactive plant compounds, known as phytochemicals, have the potential to improve health. Unfortunately, the bioavailability and bioactivity of phytochemicals such as polyphenolic flavonoids are reduced due to conjugation with sugar moieties. Here, we combine acid hydrolysis and tailored fermentation by lactic acid bacteria (Lactiplantibacillus plantarum) to convert the biologically less active flavonoid glycosides hesperidin and naringin into the more active aglycones hesperetin and naringenin. Using a comprehensive approach, we identify the most effective hydrolysis and fermentation conditions to increase the concentration of the aglycones in citrus extracts. The higher cellular transport and bioactivity of the biotransformed citrus extract are also demonstrated in vitro and in vivo. Superior antioxidant, anti-inflammatory and cell migration activities in vitro, as well as intestinal barrier protecting and antioxidant activities in Drosophila melanogaster are identified. In conclusion, the presented biotransformation approach improves the bioactivity of flavonoids, clearly traced back to the increase in aglycone content.