Alkannin is a potent pharmaceutical substance with a wide spectrum of biological activities. In the scope of this study, supercritical CO2 extraction and sonication with hexane were applied to various Alkanna species, which were then subjected to hydrolysis. Total alkannins were quantified by HPLC/DAD and incorporated into yoghurt. Viscosities, pH values and microbial analyses were reported at 7 days of intervals for 21 days of storage. A. tinctoria possessed the highest amounts of alkannins and total phenols (686.3 mg GAE/g extract). The results revealed no significant changes in pH values (4.1-4.0), viable counts of Streptococcus salivarius ssp. thermophilus (80-150 x 10(6) cfu g(-1)) and slightly lower viscosities of enriched yoghurts (8,250-6,750 cPs) compared with the control (4.15-4.0; 110-105 x 10(6) cfu g(-1); 12,600-11,310 cPs) during storage. However, viable counts of Lactobacillus delbrueckii ssp. bulgaricus of enriched yoghurts (87 x 10(3) cfu g(-1)) were much better than the control (191 x 10(3) cfu g(-1)), indicating a significant decrease in post acidification and generation of bitter peptides. Among the species investigated, A. tinctoria is the most promising source, obtained at higher yields via supercritical fluid extraction technology as a green alternative to solvent extraction and thus can be utilized at industrial scale in order to develop yoghurt products with improved health benefits.