The rising interest in finding alternatives to animal rennet in cheese production has led to studying the technological feasibility of using and exploiting new species of herbaceous plants. In this research work, and for the first time, freeze-dried extracts fromCynara humilisL. (CH) andOnopordum platylepisMurb. (OP) were studied for mineral and protein content, and their clotting and proteolytic activity were compared to those ofCynara cardunculusL. (CC). The effect of extract concentration (5–40 mg extract/mL), temperature (20–85 °C), pH (5–8), and CaCl2concentration (5–70 mM) on the milk clotting activity (MCA) of CC, CH and OP extracts was evaluated. The MCA values were significantly higher in CC at the same extract concentration. The extract that showed the most significant increase in clotting activity due to increased temperature was OP, with maximum activity at 70 °C. The pH value for maximum milk clotting was 5.0 for both CC and CH, whereas, in the case of OP, the pH value was 5.5. CaCl2enhanced the clotting capacity of the extracts, particularly for OP and CH. Furthermore, proteolytic activity (PA) and the hydrolysis rate increased with increasing time and enzyme concentration, with CC being the extract that achieved the highest caseinolytic activity.