Cheese production is an ancient practice to preserve a perishable food, such as milk, for a long time. The first step of cheese processing involves the addition of rennet, which contains the enzymes necessary for the hydrolysis and coagulation of the caseins present in milk. Typically, animal-derived rennet, such as calf rennet containing chymosin, are used as source of enzymes for cheese processing. Alternatively, microbial chymosin or recombinant chymosin is used. However, recently, plant-derived rennet such as the ones derived from thistle and bitter orange flowers and from artichoke (Cynara cardunculus var. scolymus) have also been demonstrated to be valid sources of enzymes for cheese processing. Therefore, herein, different plant and fruit extracts were tested and compared for their ability to coagulate milk caseins. In particular, beyond artichoke and cardoon (Cynara cardunculus) extracts, those from pineapple (Ananas comosus (L.) Merr.), papaya (Carica papaya L.), common fig (Ficus carica L.) milky sap, and oyster mushroom (Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm.) were investigated for their proteolytic, esterase, and milk-clotting activities. The extracts were then exploited as vegetable and fruit rennet for the experimental production of cheeses, which were examined, after 30 days of maturation, for their moisture, fat, protein, and free fatty acid (FFA) content. Interestingly, the artichoke, cardoon, and thistle mushroom extracts showed high proteolytic activity compared to calf rennet, while the level of esterase activity appeared to be similar for all the extracts. The papaya extract showed the lowest proteolytic and esterase activity. Although the pH, moisture, fat, and protein contents were very similar to those of cheese made with calf rennet, the medium- and long-chain FFAs broadly differed among produced cheeses, with variations in the lipid quality indices.