Ocimum basilicum L. (Purple basil) is a source of biologically active antioxidant compounds, particularly phenolic acids and anthocyanins. In this study, we have developed a valuable protocol for the establishment of in vitro callus cultures of O. basilicum and culture conditions for the enhanced production of distinct classes of phenylpropanoid metabolites such as hydroxycinnamic acid derivatives (caffeic acid, chicoric acid, rosmarinic acid) and anthocyanins (cyanidin and peonidin). Callus cultures were established by culturing leaf explants on Murashige and Skoog medium augmented with different concentrations of plant growth regulators (PGRs) [thidiazuron (TDZ), α-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP)] either alone or in combination with 1.0 mg/L NAA. Among all the above-mentioned PGRs, NAA at 2.5 mg/L led to the highest biomass accumulation (23.2 g/L DW), along with total phenolic (TPP; 210.7 mg/L) and flavonoid (TFP; 196.4 mg/L) production, respectively. HPLC analysis confirmed the differential accumulation of phenolic acid [caffeic acid (44.67 mg/g DW), rosmarinic acid (52.22 mg/g DW), and chicoric acid (43.89 mg/g DW)] and anthocyanins [cyanidin (16.39 mg/g DW) and peonidin (10.77 mg/g DW)] as a function of the PGRs treatment. The highest in vitro antioxidant activity was determined with the ORAC assay as compared to the FRAP assay, suggesting the prominence of the HAT over the ET-based mechanism for the antioxidant action of callus extracts. Furthermore, in vivo results illustrated the protective action of the callus extract to limit the deleterious effects of UV-induced oxidative stress, ROS/RNS production, and membrane integrity in yeast cell culture. Altogether, these results clearly demonstrated the great potential of in vitro callus of O. basilicum as a source of human health-promoting antioxidant phytochemicals.