Mulberry leaves are a well-known traditional Chinese medicine herb, and it has been observed since ancient times that leaves collected after frost have superior medicinal properties. Therefore, understanding the changes in critical metabolic components of mulberry leaves, specificallyMorus nigraL., is essential. In this study, we conducted widely targeted metabolic profiling analyses on two types of mulberry leaves, includingMorus nigraL. andMorus albaL., harvested at different times. In total, we detected over 100 compounds. After frost, 51 and 58 significantly different metabolites were identified in the leaves ofMorus nigraL. andMorus albaL., respectively. Further analysis revealed a significant difference in the effect of defrosting on the accumulation of metabolites in the two mulberries. Specifically, inMorus nigraL., the content of 1-deoxynojirimycin (1-DNJ) in leaves decreased after frost, while flavonoids peaked after the second frost. InMorus albaL., the content of DNJ increased after frost, reaching its peak one day after the second frost, whereas flavonoids primarily peaked one week before frost. In addition, an analysis of the influence of picking time on metabolite accumulation in two types of mulberry leaves demonstrated that leaves collected in the morning contained higher levels of DNJ alkaloids and flavonoids. These findings provide scientific guidance for determining the optimal harvesting time for mulberry leaves.