This study aimed to evaluate the impact of buffalo milk fresh cheese produced using M. oleifera seed milk coagulant and calf rennet. No significant difference was found between composition and functional characteristics (P > 0.05). M. oleifera seed milk coagulant cheese exhibited smaller pore size, a more uniform conformation and a denser network structure compared with calf rennet cheese. Moreover, hardness, adhesiveness, and chewiness of calf rennet cheese were significantly higher compared with M. oleifera seed milk coagulant cheese (P < 0.05). The storage modulus of both cheeses was greater than the loss modulus, thus indicating viscoelastic behavior. Moreover, the elastic gel formed in M. oleifera seed milk coagulant cheese exhibited superior stability. In addition, the content of phosphoserine, glutamic acid, long-chain fatty acids, medium-chain fatty acids, saturated fatty acids, monounsaturated fatty acids, aldehydes, esters, and lactones was significantly higher in M. oleifera seed milk coagulant cheese compared with calf rennet cheese (P < 0.05). In addition, a strong correlation was found between free amino acids, free fatty acid (FFA), and volatile flavor compounds. The findings of this study provide a theoretical foundation for the application of M. oleifera seed milk coagulant as a new plant milk coagulant resource in the dairy industry.