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In this study, a novel natural food colorant based on anthocyanins was developed from wild barberry (Berberis vulgaris L.) fruits using ultrasound-assisted extraction, which was optimized through RSM. Four extraction variables (ultrasound power, time, S/L ratio, and extraction solvent pH) were evaluated in combination. The response criteria used were the total anthocyanin content (TAC) and color parameters. The optimal TAC was achieved at 2.5 min, 345 W, pH 3, and 22.12 g/L. The fruit sample (without seeds) (BVFF) and its optimized extract (BVE) were characterized in terms of chemical composition and bioactivities. Delphinidin-3-O-glucoside was identified as the predominant anthocyanin. BVE exhibited a total phenolic content of 290.72 mg/g. Additionally, both BVFF and BVE presented significant antioxidant, antibacterial, and antifungal activity, especially in the case of BVE, which inhibited the growth of several foodborne bacteria and fungi and even showed bactericidal capacity against most of the tested bacteria, particularly against E. cloacae, E. coli, P. aeruginosa, and B. cereus. These results highlight the richness of BVFF and BVE in bioactive compounds, especially anthocyanins, underscoring their potential as natural food colorants that can be used in food product formulations instead of synthetic azo colorants.