In this study, it was aimed to develop a topically applicable nanoemulsion (NE) that is expected to have an ameliorating effect in seborrheic dermatitis (SD). The main purpose of the formulation is to eliminate the disease factor, to repair the damage caused by the disease on the skin and to smooth the skin appearance by moisturization. For this reason, in vitro antimicrobial effect and in vivo effectiveness of the formulation were tested. For this aim; essential oils from tea tree, sage, cinnamon, oregano; extracts from Aloe vera, colloidal oatmeal, liquorice; vegetable oils from grape seed and sesame, and honey were used in a NE formulation. The NEs were prepared by ultrasonication method. Preliminary stability tests were applied to all formulations and then, pH, conductivity, viscosity, average droplet size, polydispersity index (PDI), and zeta potential measurements were taken on the selected NEs for 3 months. Finally, the antimicrobial effect and in vivo effectiveness of the optimum NE were tested. The average droplet size, PDI, and zeta potential value of the optimum formulation (F6P2) were 108.40 ± 0.90 nm, 0.195 ± 0.07, and -21.40 ± 1.45 mV, respectively. As a result, the moisture content of the skin increased significantly (p < 0.001), the sebum and redness values significantly decreased (p = 0.008 and 0.001, respectively) and there was no significant change in the pH of the volunteers’ skin. Accordingly, it can be concluded that the optimum NE formulation developed in this study may be beneficial as a supplement for patients with SD.