Natural naphthoquinones such as lapachol (Lp) have demonstrated promising biological activities. However, these quinones present low systemic biodisponibility due to its poor aqueous solubility. To overcome this problem, nanoemulsions (NE) formed by oily core are able to incorporate hydrophobic molecules thus enabling them to be dispersed into an aqueous phase. The present study reports the development, characterization, and physical stability of NE containing lapachol. NE formulations (F1, F2, F3, F4, and F2-Lp) were prepared using emulsion phase inversion (EPI) method and characterized in relation to droplet size, size distribution (PDI), zeta potential, and physical stability. The formulation chosen to incorporate lapachol (F2-Lp) showed droplet size in nanometric scale with homogeneous size distribution (PDI < 0.2) and negative zeta potential (about −30 mV). In addition, good physical stability of F2 and F2-Lp was demonstrated using analytical centrifugation with photometric detection where the light transmission profiles did not change throughout the dispersions. Nanoemulsion containing lapachol presented a strong reddish aspect; however, the incorporation of this naphthoquinone did not alter the main physicochemical parameters of NE formulation. The in vitro release study demonstrated a sustained release profile from NE with about 60% of lapachol released within 54 h.