Heat-Assisted Extraction (HAE) was used for the optimized production of an extract rich in anthocyanin compounds from var. leaves. The optimization was performed using the response surface methodology employing a central composite experimental design with five-levels for each of the assessed variables. The independent variables studied were the extraction time ( , 20⁻120 min), temperature ( , 25⁻85 °C), and solvent ( , 0⁻100% of ethanol, ). Anthocyanin compounds were analysed by HPLC-DAD-ESI/MS and the extraction yields were used as response variables. Theoretical models were developed for the obtained experimental data, then the models were validated by a selected number of statistical tests, and finally, those models were used in the prediction and optimization steps. The optimal HAE conditions for the extraction of anthocyanin compounds were: = 65.37 ± 3.62 min, = 85.00 ± 1.17 °C and = 62.50 ± 4.24%, and originated 114.74 ± 0.58 TA mg/g of extract. This study highlighted the red rubin basil leaves as a promising natural matrix to extract pigmented compounds, using green solvents and reduced extraction times. The extract rich in anthocyanins also showed antimicrobial and anti-proliferative properties against four human tumor cell lines, without any toxicity on a primary porcine liver cell line.