This study was carried out with the aim of optimizing the ultrasound-assisted extraction (UAE) of phenolic compounds from male chestnut flowers (C. sativa Mill) to develop a bioactive extract with potential to be used as a natural antioxidant preservative ingredient in the food industry. Time (t, 1–39 min), solvent concentration (S, 0–100%), and ultrasonic power (P, 5–500 W) were used as the independent variables for a 5-level experimental circumscribed central composite design (CCCD) coupled with response surface methodology (RSM) to optimize the extraction of phenolic compounds by UAE. Regarding the variables, the three showed a significant effect on the extraction of phenolic compounds. The content of phenolic compounds (including flavonoids and tannins) and the extraction yield (extract weight gravimetrically assessed) were the response criteria for the optimization. Based on the statistically validated predictive polynomial models, it was possible to reach a maximum content of phenolic compounds at the global optimal conditions of 24 ± 3 min, 259 ± 16 W, and 51 ± 7% ethanol. Additionally, pentagalloyl-glucoside and trigalloyl-hexahydroxydiphenoyl-glucoside were the major phenolic compounds identified. The optimized extract was then analyzed for their biological properties. The bioactive potential of the chestnut flower extract obtained under these optimized conditions was evaluated using in vitro assays for antioxidant, anti-inflammatory, and antimicrobial activity, as well as cytotoxicity and hepatotoxicity tests. The results revealed that the enriched extract has antioxidant, antitumoral, and anti-inflammatory activities without toxicity issues. Overall, this study allowed to define the optimal conditions for the extraction of phenolic compounds from chestnuts male flowers by UAE, to obtain an enriched extract with biological properties that could be further used as a natural antioxidant ingredient with applications on functional foods.