Eucalyptus plantations in the Brazilian savanna (Cerrado) are challenged by acidic soil and water-deficit conditions. Supply of Ca and S via application of dolomitic limestone and gypsum may stimulate root development (in terms of depth) and also enhance access to water stored in the subsurface layers. Aerial and root growth, Ca2+, Mg2+, K+, and S–SO42− migration, and soil profile of Eucalyptus urophylla were evaluated following dolomitic limestone and gypsum application. Five treatments were used: (i) no application of Ca2+, Mg2+, and S–SO42− sources (CONTROL); (ii) 1200 kg ha−1 of gypsum (GYP); (iii) 2000 kg ha−1 of dolomitic limestone (DOL); (iv) DOL plus 1200 kg ha−1 of gypsum (DOL + GYP); and (v) DOL plus 2400 kg ha−1 of gypsum (DOL + 2GYP). Compared with CONTROL, Gypsum and dolomitic limestone application in DOL + 2GYP increased the concentrations of Ca2+ and Mg2+ in the soil surface and of S–SO42− in the subsurface by five, four, and three times, respectively. Fertilization with gypsum alone or in combination with dolomitic limestone did not promote an increase in fine root density (FRD) in the deeper layers. Concentrations of Ca, S, and K in the leaves increased by 16–43%. Furthermore, applying limestone 49 months after planting increased E. urophylla whole wood volume and the mean annual increment (MAI) by 8%, as compared to those in the CONTROL. Thus, gypsum and dolomitic limestone positively impacted soil fertility and may be used to acclimatize Eucalyptus to water-deficit conditions.