This study was conducted in 2020 to investigate the influence of melatonin (10, 50 and 100 mu M) given in three methods (immersion, irrigation, and foliar spraying) on some physiological aspects of tomato seedlings inoculated with root-knot nematode Meloidogyne incognita [(Kofoid and White) Chitwood]. The seedlings were inoculated with 1000 second-stage juveniles of M. incognita. Dualex((R)) optic sensor was used to in situ measure total chlorophylls, flavonols, anthocyanins contents and nitrogen balance index (NBI). Results indicated that while no significant effects were observed on chlorophyll content, melatonin ameliorated the adverse effects of M. incognita on chlorophyll depending on the concentration and mode of application. Flavonols were at the highest in the irrigated plants and the lowest in the immersed ones. NBI was affected by the method the melatonin was applied, and immersing boosted it while irrigation caused a significant decrease. Irrigation resulted in significantly higher anthocyanins compared to the other two methods. One observation was that applying melatonin in the low and medium concentrations to the soil containing nematode increased the anthocyanin content of the plant. Melatonin merits a value in developing a response against the nematode but needs further elucidation.