Insufficient exposure to light in the winter may result in a longer production periods and lower quality of seedlings in greenhouses for plug growers. Supplementary artificial lighting to plug seedlings may be one solution to this problem. The objective of this study was to assess the effects of the duration of the supplementary light on the growth and development of two watermelon cultivars, ‘Speed’ and ‘Sambok Honey’ grafted onto ‘RS-Dongjanggun’ bottle gourd rootstocks (Lagenaria siceraria Stanld). Seedlings were grown for 10 days in a glasshouse with an average daily natural light intensity of 340 μmol·m−2·s−1 photosynthetic photon flux density (PPFD) and daily supplementary lighting of 8, 12 or 16 h from mixed LEDs (W1R2B1, chip ratio of white:red:blue = 1:2:1) at a light intensity of 100 μmol·m−2·s−1 PPFD, a group without supplementary light was set as the control (CK). The culture environment in a glasshouse had 25/15 °C day/night temperatures, an 85 ± 5% relative humidity, and a natural photoperiod of 8 h. The results showed that all the growth and development parameters of seedlings grown with supplementary light were significantly greater than those without supplementary light (CK). The 12 and 16 h supplementary light resulted in greater growth and development parameters than the 8 h supplementary light did. The same trend was also found with the indexes that reflect the quality of the seedlings, such as the dry weight ratio of the shoot and root, total biomass, dry weight to height ratio of scions, and specific leaf weight. The 12 h and 16 h light supplements resulted in greater Dickson’s quality indexes compared to the 8 h supplementary light, and the 12 h supplementary light showed the greatest use efficiency of the supplementary light. 16 h of daily supplementary light significantly increased the H2O2 content and the antioxidant enzyme activities in seedlings compared to the other treatments. This indicated that 16 h of supplementary light led to certain stresses in watermelon seedlings. In conclusion, considering the energy consumption, 12 h of supplementary light was the most efficient in improving the quality of the two cultivars of grafted watermelon plug seedlings.