Skin irritation and itching, often intense, are common consequences of mosquito bites. Interest in cosmetics formulated with natural ingredients has markedly increased among consumers, perceiving them as healthier, safer and more sustainable. We developed a natural formulation based on botanical ingredients for the treatment of mosquito bites. In this work, we tested the physicochemical and microbiological stability of this novel formulation, as well as its skin tolerance. Moreover, we performed an in vitro test to assess its soothing efficacy in comparison with an ammonia-based product. According to our findings, the natural formulation resulted in a stable oil-in-water emulsion, with an appealing texture and good skin tolerance; no cases of erythema or edema were observed in any of the volunteers with sensitive skin after product application. In addition, an in vitro test performed on a keratinocytes cell line showed that our formulation significantly reduced IL-1α levels, with a displayed efficacy comparable to the ammonia-based product. This study emphasizes the promising potential of botanical ingredients in the field of cosmetic dermatology, offering high-performance, well-tolerated skincare products suitable for the treatment of common skin irritations, such as mosquito bites.