The growing demand for lettuce has prompted the need for higher quality standards. Consequently, researchers have focused their efforts on identifying cultural management strategies that can enhance the synthesis of antioxidant compounds, leading to improved functional properties of lettuce. In this regard, two experiments were conducted on hydroponically grown Lollo Bionda ‘Levistro’ and Lollo Rosso ‘Carmoli’ lettuces, known, respectively, for their green and red crispy leaves. The first experiment assessed the effects of harvest time and cutting on fresh weight (FW), dry weight (DW), total phenolic content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC), and antioxidant capacity (AC). The second experiment evaluated the response of FW, DW, TPC, TFC, TAC, AC, proline content, and morphological cell changes to UV-B supplementation in greenhouse conditions as well as the impact of successive harvests on the same plant. UV-B radiation and cutting led to a reduction in FW, but they also showed an increase in DW. Furthermore, UV-B radiation, cutting, and plant growth stage had significant effects on TPC, TFC, and AC in both cultivars. Applying 10.5 kJ m−2 of UV-B radiation or performing different harvests resulted in increased TFC in ‘Levistro’, exhibiting a remarkable 91% increase at the third harvest compared to the control group (0 kJ m−2 at the first harvest). UV-B radiation also induced changes in anatomical cell distribution in both cultivars, leading to a 37% increase in intracellular space in ‘Levistro’ and a reduction of up to 8.2% in ‘Carmoli’. Lastly, at a later stage of plant development (9-10th true leaves), ‘Carmoli’ demonstrated a 51% increase in TPC, 95% in TFC, and 65% in TAC, highlighting its potential as an intriguing strategy to obtain lettuce varieties with higher antioxidant properties. These findings underscore the significance of implementing cultural management techniques to enhance the antioxidant composition of lettuce.