Iron is a fundamental element for plants as well as for microorganisms and several pathogens, including Rhizoctonia solani, one of the main soil-borne pathogens of tomato. This study demonstrated the ability of Aureobasidium pullulans strain L1 to produce siderophores and how these molecules were, directly and indirectly, connected to its antagonistic activity and iron bioavailability. By ICP-OES (Inductively Coupled Plasma-Optical Emission Spectroscopy) trace analysis, the strain displayed the ability to increase the bioavailability of Fe (II) in the soil by almost 50% 30 days after inoculation. Also, the bioavailability of Mn, Cu and Zn was increased after 30 days of incubation in the soil by 31.8, 38.4 and 27.1%, respectively. In in vivo assays, A. pullulans L1 strain showed a growth promotion of tomato roots length and stem diameter, respectively, by 19.1 and 27.3%, and acted as a biocontrol agent (BCA) against R. solani (80% of inhibition). The results demonstrate a new aspect of this microorganism, usually applied as an antagonist of postharvest fruit diseases, to explore in different environments and against different pathogens, such as soil-borne pathogens.