This study aimed to determine the feasibility of temperature difference as an overgrowth-prevention technique to influence plant height and internode length in a plant factory with artificial lighting. The control plants were grown in a commercial nursery greenhouse using a growth regulator (Binnari), and +DIF (25 °C/15 °C), 0DIF (20 °C/20 °C), and −DIF (15 °C/25 °C) were the treatments with different day/night temperatures and the same average temperature (20 °C). Cucumbers showed the strongest suppression under the −DIF treatment, with a dwarfism rate of 33.3%. Similarly, tomatoes showed 0.8% and 22.2% inhibition in the 0DIF and −DIF treatments, respectively. The FV/FM of cucumber was approximately 0.81 for all treatments. The OJIP changes differed for cucumbers; however, both cucumbers and tomatoes had similar OJIP curve patterns and no abnormalities. The relative growth rate of cucumbers at the growth stage was 1.48 cm·cm·day−1 for days 6–9 in +DIF stage 3, which was the highest growth rate among all treatments, and 0.71 cm·cm·day−1 for days 3–6 in −DIF stage 1, which was the most growth-inhibited treatment. In tomatoes, we found that days 3–6 of −DIF stage 1 had the most growth inhibition at 0.45 cm·cm·day−1. For cucumber, −DIF days 3–6 had the most growth inhibition, with a relative growth rate of 0.71 cm·cm·day−1, but the fidelity was significantly higher than the other treatments, with a 171% increase. The same was true for tomatoes, with days 3–6 of −DIF stage 1 showing the most inhibited growth at 0.45 cm·cm·day−1 but a 200% increase in fidelity. Therefore, applying the −DIF treatment at the beginning of growth would be most effective for both cucumbers and tomatoes to prevent overgrowth through the DIF in a plant factory with artificial lighting because it does not interfere with the seedling physiology and slows down the growth and development stage.