Rubia tinctorum L. roots are a valuable source of anthraquinones and other bioactive phytochemicals. In this study, nine hydrophilic deep eutectic solvents (DESs), based on choline chloride or tetrabutylammonium bromide, were evaluated as green extractants for the selective recovery of bioactive compounds. Their performance was compared with four conventional solvents (acetonitrile, methanol, ethanol–water, and water). Among DESs, choline chloride–lactic acid and choline chloride–malonic acid showed the highest extraction efficiency for gallic acid, caffeine, quercetin, alizarin, and purpurin. Extraction conditions were optimized using response surface methodology. The antioxidant, antimicrobial, and phytochemical profiles of the resulting extracts were comprehensively evaluated. GC-MS analysis revealed the presence of key metabolites including alizarin, rubiadin, purpurin, scillarenin, and fucosterol. The results demonstrate the potential of hydrophilic DESs as sustainable, efficient, and selective media for the extraction of biologically active compounds from plant materials.