Hylocereus costaricensis peel contains large amounts of betacyanins and can be exploited as a source of natural colorants. This work aimed the chemical characterization and evaluation of bioactive properties of this by-product and the optimization of the ultrasound-assisted extraction (UAE) of betacyanins using the response surface methodology (RSM). Oxalic and malic acids and traces of fumaric acid were detected, as well as the four tocopherol isoforms, predominantly γ-tocopherol. Four betacyanins were identified and used as response criteria for UAE optimization, namely phyllocactin, isobetanin, isophyllocactin, and betanin. Sample processing at 487 W for 38 min result in the maximum betacyanin content (36 ± 1 mg/g dw). The peel extract inhibit the oxidative haemolysis, with IC₅₀ values of 255 and 381 μg/mL for Δt of 60 and 120 min, respectively, and the growth of pathogenic bacteria, with minimum inhibitory concentrations ranging from 5 to 20 mg/mL. Furthermore, no toxicity was observed for normal cells.