This study investigated the impact of variety and harvest time on the visual appearance, nutritional quality, and functional active substances of six lotus root cultivars: “Xinsanwu”, “Wuzhi No. 2”, “Baiyuzhan”, “Huaqilian”, “Elian No. 6”, and “Elian No. 5”. Samples were collected monthly from December 2023 to April 2024. A nutrient analysis revealed a decrease in the water content with a delayed harvest. The total soluble solids and soluble sugar content peaked towards the end and middle-to-late harvest periods, respectively. Starch levels initially increased before declining, while the soluble protein exhibited a triphasic trend with an initial rise, a dip, and a final increase. The vitamin C (Vc) content varied across cultivars. Functional active substances displayed dynamic changes. The total phenolics initially decreased, then increased, before ultimately declining again. The total flavonoid content varied by both cultivar and harvest time. The phenolic acid and flavonoid content mirrored the trends observed for total phenolics and total flavonoids. Gastrodin was the most abundant non-flavonoid compound across all varieties. “Wuzhi No. 2” and “Baiyuzhan” displayed higher levels of functional active substances and starch, while the Elian series and “Xinsanwu” cultivar exhibited a greater content of Vc, soluble sugar, and soluble protein. Specific harvest periods yielded optimal results: “Wuzhi No. 2” (H1 and H5), “Huaqilian” (H2), and “Baiyuzhan” (H3 and H4) demonstrated a high nutrient and functional active substance content. Overall, the lotus roots harvested in period H4 achieved the highest score. Overall, this study provides the foothold for the rapid identification of superior lotus root cultivars and the valorization of lotus root by-products via advanced processing methods. Additionally, it offers valuable insights for market participants and consumers to select optimal varieties and harvest times based on their specific needs.