Sweet basil (Ocimum basilicum L.; Fam. Lamiaceae) is an annual herbaceous plant with a high economic value used in folk medicine, pharmacology, and food production. In Italy, most of the varieties are used to produce the famous “pesto” sauce; however, almost all of them are susceptible to basil downy mildew (BDM) disease, strongly decreasing the growth of the fresh leaves and the survival of the whole plant. Nowadays, CRISPR/Cas9 technology is recognized to be a prominent way to enhance basil genetic breeding. In this work, we present an optimized protocol for in vitro direct regeneration of an elite cultivar, which is the major limiting factor for the transformation of O. basilicum. Regeneration has been obtained from different explants (leaves, cotyledons, cotyledonary nodes); the highest frequency has been obtained from cotyledonary nodes of seedlings germinated on MS medium containing TDZ. This protocol may be used for biotechnological applications as genome editing techniques to obtain basil-downy-mildew-disease-resistant clones.