The aim of this study was to determine the best time to harvest mulberry (Morus alba L.) leaves for optimal yields of functional components. Mulberry leaves were analyzed to determine the seasonal changes in the concentrations of functional components, including flavonols, chlorogenic acid, and 1-deoxynojirimycine (DNJ). During the experimental period (May 26–October 16), the ranges of flavonols, chlorogenic acid, and DNJ contents were 1134–2230 mg/100 g dry weight (DW), 616–1014 mg/100 g DW, and 53–199 mg/100 g DW, respectively. There were high flavonols contents in mulberry leaves from late May to early July, from early to mid-August, and after late September. There were high chlorogenic acid contents from late May to early July, and after late September. From late September, the flavonols contents increased over time, and showed the highest level on October 16, the last day of the experimental period. The increased flavonols contents after late September were due to the synergistic effects of solar radiation and lower temperatures. The DNJ content showed a bell-shaped curve, peaking in mid-August, and the DNJ content was strongly correlated with total temperature. The flavonols and chlorogenic acid contents in mulberry leaves were higher after late September when the temperatures decreased and there was still high solar radiation, while the DNJ content peaked in August when temperatures were highest. The results indicated that August is the best time to harvest mulberry leaves to obtain optimal yields of beneficial functional components. If mulberry leaves are harvested twice per year, then the first harvest should be in July to August when temperatures and solar radiation hours are high, and the second harvest should be completed by the end of September.