The bioactive compound, thymoquinone (TQ), found in Nigella sativa seeds is known for its various therapeutic properties. Thymoquinone (TQ), a bioactive compound found in Nigella sativa seeds, is known for its various therapeutic properties. However, TQ as a compound is challenging for a pharmaceutical formulator due to its hydrophobic nature and chemical instability, limiting its skin penetration. Transethosomes, as a formulation, provide an environment that enhances the solubility of TQ, stability, as well as skin permeation. To optimize TQ transethosomal formulations, we introduced a combination of surfactants namely nonionic Tween 20 and ionic sodium lauryl sulfate (SLS) or sodium lauroyl glutamate (SLG). These surfactants stabilize the formulation, reduce aggregation, and improve biocompatibility, as well as minimizing potential toxicity. Using the Langmuir monolayer technique, we fine-tuned the formulation composition and gained insights into its interfacial behavior. We conducted skin penetration studies using Rhodamine B as a model lipophilic compound to understand the skin penetration mechanisms. Our findings revealed that the transethosomes formulation, a combination of ionic and nonionic surfactants in the F2 formulation, resulted in the smallest particle sizes (115.4 nm) and demonstrated the highest skin permeation after 24 hours (267.13 μg/cm²), surpassing the control and ethosomal formulations. Additionally, this transethosomal formulation exhibited antimicrobial activity against S. aureus, with a zone of inhibition of 26.4 ± 0.3 mm. Importantly, we assessed the cytotoxicity of both ethosomes and transethosomes at concentrations ranging from 3.5 μM to 50 μM on HaCaT cell lines and found no cytotoxic effects in comparison to TQ solution controls. These results suggest the potential safety and efficacy of these TQ formulations and their further development in skin delivery.