Edible flowers have been used as ingredients because of their biological activities, taste, and overall appearance. This research was aimed to characterize the chemical composition and in vitro antioxidant activity of the marigold flower (Calendula officinalis L.) extracted with different proportions of water and ethyl alcohol, and the lyophilized extract with higher content of antioxidant compounds was incorporated into an organic yogurt. Results showed that the hydroalcoholic extract (50:50 v/v) presented the highest total phenolic content (TPC), flavonoids, and antioxidant activity (ferric reducing antioxidant power (FRAP), total reducing capacity (TRC), and Cu2+/Fe2+ chelating ability). Phenolic acids and flavonoids were quantified in the extract by LC-DAD, while 19 compounds were tentatively identified by ESI-MS/MS. The lyophilized marigold extract (LME) also inhibited 12% of Wistar rat’s brain lipid oxidation in vitro, inhibited α-amylase, and α-glucosidase activities, but showed no cytotoxicity towards cancerous cells (HCT8 and A549). However, marigold flower extract protected human erythrocytes against mechanical stress. When added into an organic yogurt model (0 to 1.5%), LME increased TPC and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH) and TRC), and the sensory analysis showed that the organic yogurt had an acceptance of 80.4%. Our results show that the use of LME may be a technological strategy to increase the content of bioactive compounds in yogurts.