Astragalus membranaceus Bunge and Codonopsis lanceolata Benth. et Hook. f. are two famous medical species in Korea, China, and Japan, mainly used for treating diseases including cancer, obesity, and inflammation. Manipulation of the difference between the day and night temperatures (DIF) is an efficient horticultural practice to regulate the growth and development of vegetables in a glasshouse. However, little research has focused on how the DIF influences the plug seedling quality of medicinal plants. In this study, uniform plug seedlings were cultivated in three environmentally controlled chambers under an average daily temperature of 20 °C with negative (−10 °C), zero, or positive (+10 °C) DIFs, and the same relative humidity (75%), photoperiod (12 h), and light intensity (150 μmol·m−2·s−1 photosynthetic photon flux density with white LEDs). The results showed that the DIF had a noticeable effect on the growth, development, and morphology of A. membranaceus and C. lanceolata plug seedlings. The positive DIF (+10 °C) significantly increased the biomass (shoot, root, and leaf), stem diameter, and Dickson’s quality index, indicating an enhanced plug seedling quality. Moreover, the contents of primary and secondary metabolites, including soluble sugar, starch, total phenols and flavonoids, were higher with higher DIFs, where the maximum values were found at 0 °C or +10 °C DIF. Furthermore, the increases in the chlorophyll content and stomatal conductance were obtained in a positive DIF, indicating that a positive DIF was favorable to photosynthesis. An analysis of the gene expression showed that a positive DIF (+10 °C) up-regulated the expression of photosynthetic genes, including GBSS, RBCL, and FDX. In conclusion, the results of this study recommend a positive DIF (+10 °C) for enhancing the quality of A. membranaceus and C. lanceolata plug seedlings.