Black elderberry (Sambucus nigra L.) flowers are rich in polyphenolic compounds, including chlorogenic acid and quercetin derivatives, which are known for their health benefits, particularly antioxidant and antidiabetic properties. This study aimed to optimize the extraction conditions using the Box-Behnken model to maximize polyphenol yields from different elderberry flower varieties and to evaluate their potential for antidiabetic action. The extracts were analyzed for their phytochemical content and assessed for enzyme inhibition, specifically targeting enzymes critical in carbohydrate digestion and glucose regulation. The anti-inflammatory activity was also assessed. Results indicated that the Black Beauty, Obelisk, and Haschberg varieties demonstrated significant inhibition of α-glucosidase, with a high inhibitory potential against α-amylase enzymes for Obelisk variety. Additionally, high chlorogenic acid content strongly correlated with enzyme inhibition and antioxidant activity, suggesting its substantial role in glucose regulation. The study underscores the potential of elderberry flower extracts, particularly those rich in chlorogenic acid, as natural agents for managing blood glucose levels, warranting further exploration of their use in antidiabetic applications.