Utilization of nitrogen (N) element is a common practice used to reach profitable yields in horticultural crops and N supply can be used as a tool to manipulate the enhancement of phytochemicals and minerals in vegetable crops to address consumer-oriented quality production.  Hence the study was aimed to investigate the effect of N application on leaf morphology, ascorbic acid content, phenolic acids, flavonoids, mineral content, and nitrate residues in three lettuce varieties (two green leafy lettuce, Multigreen 1 and Multigreen 3; one red leafy lettuce, Multired 4) grown in a closed hydroponic system (gravel-film) at harvest.  Nitrogen was applied as ammonium nitrate (NH₄NO₃) at six different concentrations of 0, 60, 90, 120, 150 and 180 mg L^–1.  The results obtained during 2015 and 2016 seasons were similar and demonstrated variety dependent responses with respect to different N application rates.  Multigreen 3 was more sensitive to N supply and showed higher amount of nitrate residue at harvest.  Variety Multired 4 was less sensitive to N supply followed by Multigreen 1.  Although N supply at 120 mg L^–1 improved the yield and the number of leaves in Multigreen 3, overall 90 mg L^–1 can be recommended for these lettuce varieties to improve the yield and the accumulation of ascorbic acid content, phenolic acids mainly caffeic, caftaric acids, quercetin (the important flavonoid in lettuce), and Fe and Mn contents.  Furthermore, the concentration of 90 mg L^–1 improved the antioxidant property (FRAP and ABTS+) and reduced the nitrate accumulation, ensuring safe food for consumers.