The purpose of this work was to analyse the influence of rennet from different Cynara cardunculus plants, selected for its clotting and proteolytic activity on caseins, on the characteristics of manufactured ‘Torta del Casar’ cheeses. After classifying the cardoon according to proteolytic activity into five groups of greater or lesser activity, 16 batches of cheeses were made with rennet derived from different wild cardoon plants. We observed a major development of the proteolysis during ripening leading to the generation of non-protein nitrogen compounds. Especially noteworthy was the relationship of amino acid nitrogen (AN) generation with rennet clotting activity after 24 h of maceration, and the fact that the production of biogenic amines was not related to the proteolytic activity of the rennet. The activities of the rennet observed ‘in vitro’ were also developed ‘in vivo’ in the cheeses, with the different rennets used affecting the final sensory characteristics of cheeses. The rennet with high clotting activity after 24 h of maceration was positively correlated with the creaminess, viscosity, and acceptability of the cheese. However, the high proteolytic activity rennet negatively influenced the acidity, bitterness, and creaminess parameters. Therefore the most appropriate cardoons for making this cheese are those with higher clotting activities and moderate proteolytic activities especially on β-casein. The use of controlled and characterised cardoons in the manufacturing process of Torta del Casar is fundamental to obtaining the homogeneous product demanded by the Torta del Casar Registry of the Protected Designation of Origin.