Basil (Ocimum basilicum L.) is an essential ingredient of the Mediterranean cuisine due to its distinctive aroma. Genovese basil leaves are used to prepare “pesto”, a condiment that has always caught the interest of consumers and producers. Usually, basil for industrial processing is harvested more than once to extract a higher yield. However, successive cuts can affect quality traits that play a crucial role in defining the product’s final sensory profile. This research was aimed to evaluate the impact of cut on the quantitative and qualitative properties of three Genovese basil cultivars (Aroma 2, Eleonora and Italiano Classico) grown in an open field. Nitrate content, phenolic acids and aromatic profile were determined by ion chromatography (IC), high-performance liquid chromatography (HPLC), and gas chromatography coupled to a mass spectrometer (GC/MS) analysis, respectively. The second harvest increased fresh biomass and total phenolic acids content by 172% and 413%, respectively, with Italiano Classico recording the highest values. The combination of second-cut Aroma 2 yielded the lowest nitrate (473.8 mg kg−1 of fresh weight) and Eugenol (2.4%) levels. In the second harvest, Eleonora showed an increase in eugenol and trans-α-bergamotene of 75.3% and 48.2%, respectively; whereas, eucalyptol and β-cis-ocimene decreased by 34.4% and 51.6%, respectively. Although successive harvests may increase basil yield and quality overall, the cultivar-dependent response to successive cuts needs to be accounted for in order to accomplish standardization of industrial “pesto” sauce.