Repurposing saffron (Crocus sativus) waste presents a sustainable strategy for generating high-value products within the bioeconomy framework. Typically, flower components are discarded after stigma harvest, resulting in significant waste—350 kg of tepals per kilogram of stigmas. This research employed a comprehensive approach, integrating bioactivity studies (in vitro and in silico) with Life Cycle Assessment (LCA) evaluations, to extract and assess bioactive compounds from C. sativus tepals sourced in Tuscany, Italy. Phytochemical characterization using UPLC-MS/MS revealed a high abundance and variety of flavonoids in the hydro-ethanolic extract (CST). The antioxidant capacity was validated through various assays, and the ability to mitigate H₂O₂-induced oxidative stress and enhance fermentation was demonstrated in Saccharomyces cerevisiae. This study reports that C. sativus tepals extract reduces oxidative stress and boosts ethanol fermentation in yeast, paving the way for applications in the food and biofuels sectors. Further validation in RAW 264.7 macrophages confirmed CST’s significant anti-inflammatory effects, indicating its potential for pharmaceutical, cosmeceutical, and nutraceutical applications. In silico studies identified potential targets involved in antioxidant and anti-inflammatory processes, shedding light on possible interaction mechanisms with Kaempferol 3-O-sophoroside (KOS-3), the predominant compound in the extract. The integration of LCA studies highlighted the environmental benefits of this approach. Overall, this research underscores the value of using waste-derived extracts through “green” methodologies, offering a model that may provide significant advantages for further evaluations compared to traditional methodologies and supporting the circular bioeconomy.