Melatonin has been recognized as a regulator of plant growth and development. The present study aims to optimize the appropriate dosage of melatonin through seed priming, taking into account a range of physio-biochemical traits. Two distinct rice genotypes with divergent yield potentials, namely N-22 (drought-tolerant) and IR-64 (drought-susceptible) were subjected to priming with varying concentrations of melatonin (0, 60, 90, 120, 150, and 180 ppm). Our study revealed that, at the 120-ppm melatonin concentration, IR-64 demonstrated superior germination rates, root biomass, shoot biomass, chlorophyll content, and total antioxidant activity as compared to N-22 genotype. Conversely, N-22 displayed enhanced shoot length and root length in contrast to IR-64, at the 120-ppm dosage. Additionally, the melatonin concentration of 120 ppm exhibited profound efficacy in reshaping root-shoot architecture (RSA) in both genotypes. Consequently, the optimal melatonin dosage for effective seed priming was established as 120 ppm. Further, priming the seeds of IR-64 and N-22 with melatonin (120 ppm) holds the potential to enhance rice yield through the augmentation of overall growth and development. These findings enrich our comprehension of melatonin's influence on rice seed priming and pave the path for future explorations into the fundamental mechanisms governing these effects.