Betalains are accepted food additives derived from vacuoles of plants belonging to about 17 families in the order Caryophyllales. These pigments are composed of a nitrogenous core structure, betalamic acid [4-(2-oxoethylidene)-1,2,3,4-tetrahydropyridine-2,6-dicarboxylic acid]. Betalamic acid condenses with imino compounds (cyclo-DOPA and/or its glucosyl derivatives) or amines and/or their derivatives to form violet betacyanins (for example, betanin) and yellow betaxanthins (for example, indicaxanthin), respectively. Till date, structures of 75 betalains have been elucidated from plants under the order Caryophyllales. The extracted betalains are safe to consume and they act as micronutrients in the body. In vitro studies to highlight radical-scavenging activity, cell culture studies to assess cytotoxicity and absorption of betalains, and proven clinical efficacies are compiled in this review. The literature on biological activity has not been analyzed for a synthesis of safety, clinical efficacy, and bioavailability to arrive at the concentrations required for the purported health benefits. Most betalains are under-utilized in pharmaceutical and cosmetic preparations due to poor stability and lack of scientific reports highlighting their superior tinctorial strength including flourescence, water solubility, and functional value alongside their bioavailability. This is the first comprehensive review on the dietary safety, biological activity and bioavailability of betalains. Based on this review, for future debate and input from health professionals, a human daily intake of betanin and indicaxanthin can be proposed at 100 and 50 mg, respectively.