The present study investigated how auxin concentration and the method of application affected the formation of adventitious roots in microshoots of chestnut (Castanea sativa) and oak (Quercus robur). The activity of two urea derivatives (2, 3-MDPU and 3, 4-MDPU) was also evaluated. Microshoots were derived from basal sprouts of two mature chestnut trees (P1 and P2) and one adult oak genotype (Sainza). In chestnut, rooting percentage was positively affected by auxin in a dose-dependent manner, particularly in shoots treated with the hormone for 24 h. The effect of auxin on rooting also differed depending on the application method. In shoots treated for 24 h, the highest concentration of auxin produced the healthiest rooted plantlets, in terms of the root system and shoot quality. By contrast, in shoots treated by the basal quick-dip method, the shoot quality was best at the lowest auxin concentration. The effect of urea derivatives on the root system depended on the species as well as on the auxin concentration and application period. Use of the MDPUs improved the root system architecture of auxin-treated shoots by promoting lateral root development and triggering the synchronous initiation of root primordia at the base of the shoot. Shoot quality was also improved by MDPUs, which promoted resumption of growth and reduced shoot-tip necrosis.