Iturin A is a very important cyclic lipopeptide produced by several B. subtilis strains and has large commercial and therapeutic application potentials but its production on industrial scale has not been realized yet. In the present study, we have observed that the strain ET-1 of Bacillus subtilis, a producer of Iturin A, can present at least three different colony morphologies, which we arbitrarily called Rough, Smooth, and Mucoid morphotypes (R-, S-, and M-form). Performing HPLC analysis, a significant difference between the amounts of Iturin A produced by the three morphotypes was found. The morphotype R-form showed the highest productivity with yields about 10 and 100 times higher than morphotypes S and M, respectively. The results show that the production of Iturin A by B. subtilis could be strongly influenced by the phenotypic heterogeneity of cells within the inoculum. Indeed, we have observed that, pasteurizing the inoculum before seeding in order to improve the homogeneity removing the phenotypes less able to synthesize the Iturin A, its yields in a bench-scale production could be significantly improved. This can represent an important control factor also at industrial scale to improve the Iturin A yields, the robustness, the replicability, and consequently the cost-effectiveness of fermentation processes.