The aim of this study was characterized the physiochemical and phytochemical properties of betalains extracted from red beetroot as antioxidant, antibacterial, and anticancer activity. Betalains were extracted from red beetroot using different single or mixed solvents in comparison with ultrasonic-assisted extraction (UAE) using different conditions from solid to solvent-ratio and duration of extraction. The content of total phenolics (TP) and flavonoids (TF), color characteristics (CIE L* a* b*) and HPLC profile were analyzed in betalains extracted under optimized conditions. Among different tested media, 0.1% ascorbic acid at 1:30 (g/ml)/ 10min and 20% ethanol acidified with 0.1% ascorbic acid at 1:15 (g/ml)/2.5 min significantly (p ≤ 0.05) exhibited higher betalains content of 1085.80 and 1760.00 mg/100gm, respectively, compared to 1662.50 mg/100g when ultrasonic assisted extraction was applied at 1:30 (g/ml)/ 10min at 40оC. High content of TP and TF as well as antioxidant activity (ABTS and DPPH) were found in the betalains recovered with optimal conditions (1:15(g/ml)/ 2.5 min). Betalains extracted was effective as antimicrobial against most of the tested microorganisms as well as a high anticancer activity against MCF-7 than Caco-2 and HepG2 cancer cells. Betalains can be recommended for the application as natural food colorants and bioactive ingredients in the food industry.