The versatile roles of D-amino acids (D-AAs) in foods, diseases, and organisms, etc., have been widely reported. They have been regarded, not only as biomarkers of diseases but also as regulators of the physiological function of organisms. Over the past few decades, increasing data has revealed that D-AAs have great potential in treating disease. D-AAs also showed overwhelming success in disengaging biofilm, which might provide promise to inhibit microbial infection. Moreover, it can effectively restrain the growth of cancer cells. Herein, we reviewed recent reports on the potential of D-AAs as therapeutic agents for treating neurological disease or tissue/organ injury, ameliorating reproduction function, preventing biofilm infection, and inhibiting cancer cell growth. Additionally, we also reviewed the potential application of D-AAs in drug modification, such as improving biostability and efficiency, which has a better effect on therapy or diagnosis.