The present study deals with the control of two important postharvest fruit diseases by application of the supernatant obtained from Bacillus subtilis strains ET-1. The bacteria, B. subtilis was grown in a bench-scale bioreactor and a cell free supernatant (CFS) rich in Iturin A was produced. During the entire bacterial culture time, it has been observed that Iturin A production started at the end of the exponential growth phase and a maximum concentration of 422 mg L−1 was observed after 57 h. CFS was then used to evaluate, both in vitro and in vivo, for its antifungal activity against P. digitatum and B. cinerea the agents responsible for the green and gray mold, respectively. Concerning in vitro studies, it has been observed that the antifungal activity of CFS was correlated to Iturin A concentration. The minimum concentration of Iturin A within CFS, necessary to inhibit P. digitatum and B. cinerea, was 6.60 and 3.30 mg L−1, respectively. The application of CFS on lemon and strawberry fruit artificially inoculated with P. digitatum and B. cinerea proved to be highly effective in the control of rot diseases. Compared to the positive control, in the CFS treated trials a significant decrease of disease incidence (up to 68.6% and 74.1%, in lemon and strawberry respectively) was recorded. It is also important to emphasize that the CFS treatment has drastically prevented the expansion of the fungal mycelium on the diseased fruits. The strong antifungal activity together with its already known characteristics related to high biodegradability and low toxicity, suggest that Iturin A could be a successful alternative to synthetic fungicide usage in postharvest disease control. However, it is important to do further research on large scale basis to evaluate the Iturin A production and extraction by B. subtilis ET-1 in order to study the possible formulations for its ideal application.