We investigate the impact of plasma activated water (PAW) on the root development of Brassica juncea using a phytofluidic device. We prepared PAW with an adjusted pH level employing a state-of-the-art microbubble enhanced cold plasma activation (MB-CPA) technique. The results manifest that the root length and the number of cortical cells increases as the percentage of PAW rises up to 20%, attributed primarily to the enhancing nitrogen (N)-uptake trend. Whereas, beyond ∼20% PAW concentration, a limited root development (30 to 60% reduction) was observed due to the adverse effect of excess nitrate or nitrite ions of PAW, generating greater oxidative stress. Consequently, we obtain two distinct sets of Michaelis-Menten kinetics parameters for two N-uptake regimes in the window of PAW percentage under consideration. The higher magnitude of inward diffusive flux of N-uptake is evident at lower PAW percentages and lower at larger PAW percentages. Intriguingly, at a PAW percentage closer to 20%, the pectin’s Raman peak intensity reaches its maximum value for cell signaling of nutrition transport. Similarly, the higher Young’s modulus value for 20% PAW permits greater mechanical strength because of the enhanced lutein intensity. Besides, we performed numerical simulations of the flow field developed inside the device, and the simulated results also confirm that the mechanical stress at the root tip region is significantly reduced on the application of PAW. The inferences drawn from this analysis offer insights into how PAW influences plant-root development in sustainable agricultural techniques, including hydroponic systems.