Purpose <p>This study provides insights that blessed thistle (C<i>nicus benedictus</i> L.) ecotypes grown in different eco-edaphic conditions can be targeted as a potential source of health-relevant phytochemicals linked to their antioxidant and enzymatic inhibitory activities.</p> Methods <p>Agronomic productivity, phytochemical contents as well as cnicin concentration and phenolic profile linked to the functional biological properties (α-amylase and α-glucosidase inhibitory and antioxidant activities) were assessed with three ecotypes (Tehran, Shiraz, Sari) grown in four various eco-edaphic sites (S1-S4) during two growing seasons.</p> Results <p>The greatest biomass yield (12,005&#xa0;kg DW ha^− 1) was noted for plants grown in the arid-sandy soil condition (S1), while the lowest yield (10,357&#xa0;kg DW ha^− 1) was recorded for plants grown in the humid climate-clay soil condition (S4). The Tehran ecotype demonstrated richer phytochemicals in arid and semi-arid sites compared to others. The HPLC analysis showed that while chlorogenic acid, as the dominant phenolic compound, was higher in ecotypes grown in S1; quercetin and rutin, the minor compounds responsible for plant enzymatic inhibitory, were more abundant in the semi-arid (S2) plants.</p> Conclusions <p>Cnicin and individual phenolic acids like chlorogenic acid and flavonoids, mainly quercetin and rutin, had a potential synergetic effect on the plant’s amylase and α-glucosidase inhibition potential. The Tehran ecotype could be considered the most important therapeutically relevant ecotype for future plant variety developments. The high agronomic productivity of blessed thistle in marginal lands and its biological activity linked to certain phytochemicals make the plant a potential target for further developments and glycemic control therapeutic applications.</p>