Osmoporation is a novel encapsulation approach for bioactive compounds based on the osmoresistance mechanisms of microbial cells. To the best of our knowledge, this is the first study investigating the production of fisetin-enriched yoghurt using Lactobacillus acidophilus-based bio-capsules via osmoporation as the starter culture. Results showed that the milk acidification with fisetin-loaded L. acidophilus progressed at a slower pace due to complex mechanisms induced by osmoporation and internalized fisetin. Milk fermentation using fisetin bio-capsules reached a maximum acidification rate of 0.18 pH units/h after 23 h and pH 4.6 was achieved after 32 h. Besides, the antioxidant activity of yoghurts produced with fisetin bio-capsules did not change during cold storage, while the antioxidant activity of yoghurt produced with non-encapsulated fisetin was reduced by 2.5-fold after 28 days. Overall, this study shows that fisetin osmoporation using L. acidophilus is a versatile encapsulation bioprocess that enables the delivery of preserved phytoactives into fermented foods like yoghurt. This strategy has the potential to be extended to other applications in the dairy industry using lactic acid bacteria as both the encapsulation matrix and fermentation agent.