To investigate the effect of herbicide Trifluralin and the role of magnetized water on enzymatic activities and content of proline and growth substances including gibberellin (GA₃) and cytokinin (6- benzyl amino purine), an experiment was conducted using squash (Cucurbita pepo L. var. Shiraz Hybrid F1) seedlings. Four levels of trifluralin (0, 5, 15 and 25 ppm) and irrigation by distilled and magnetized water were applied in a completely randomized design in 3 replicates at Urmia University in 2016. Different levels of trifluralin were added to pots 2 days after planting. Seedlings were treated by modified Hoagland solution with diurnal temperature 30:18 °C, relative humidity of 70-80 percent and light period of 16:8 hr, during a 5-week growing period in growth chamber. The effect of magnetized water and trifluralin on ornithine amino-transferase (OAT), proline dehydrogenase (PDH) and content of proline in both shoots and roots as well as GA₃ and cytokinin was significant (p>0.01) according to ANOVA. The content of proline and OAT in roots and shoots as well as GA₃ and cytokinin increased significantly, although the PDH of roots and shoots decreased under the influence of magnetized water. The content of proline and OAT increased and GA₃, cytokinin and the activity of PDH declined by the increase of the levels of trifluralin. Overall, it can be concluded that the growth and biochemical indices of squash plants were improved by magnetized water under toxicity of herbicide trifluralin.