Shoots of the olive cultivar Moraiolo were previously cultured in aseptic conditions on Olive Medium (OM), with the addition of 4 mg·L−1 of zeatin, 30 g·L−1 of sucrose, and 7 g·L−1 of agar. Then, 1-cm long uninodal explants with two leaves and two axillary buds were excised from the proliferated masses and placed on the same proliferation medium enriched with four concentrations of neem oil (0—control, 0.1, 0.5, and 1.0 mL·L−1), added before sterilization. The addition of 0.1 mL·L−1 of neem oil to the medium gave an improvement in shoot regeneration. More vigorous shoots (longer proliferated shoots) were obtained along with a higher number of nodes (multiplication rate). Overall, there was a significant increase in the total fresh and dry proliferated weights. To our knowledge, this is the first report showing a strong and beneficial effect of neem oil, used as a “complex mixture,” on in vitro plant regeneration.