Background: Carvacrol (CAR) is the active component in essential oils (EOs) of many fragrant plants, including oregano and thyme; however, its high toxicity restricts its usage in biomedical fields, and the self-nano-emulsifying drug delivery system (SNEDDS) was suggested to overcome this issue. Objective: To prepare and characterize the CAR-loaded SNEDDS and to assess its toxicity profile towards healthy rats using the in vivo sub-chronic study. Methods: SNEDDS was prepared from olive oil, dimethyl sulfoxide, Tween-80, and distilled water, then CAR-SNEDDS was prepared by adding 0.5% CAR to SNEDDS and both composites were gently agitated for 72 h at room temperature. Later on, both composites were physiochemically characterized for size/charge (Zetasizer), shape (TEM), crystallinity (XRAD), composition (FTIR), and quantitated (UV–Vis). Additionally, the sub-chronic toxicity of both composites at different doses was conducted by orally treating healthy Sprague Dawley for 4 weeks. Then, the treated rats were checked for toxicological symptoms, food/water intake, and behavioral abnormality. In addition, the blood samples were tested for hematologic/biochemical changes, while vital organs (liver and kidney) were assessed for histopathological alterations. Results: The average globule size, zeta potential, and polydispersity index of CAR-SNEDDS were 158.93 ± 22.18 nm, −22.56 ± 1.77 mV, 0.553 ± 0.31, respectively. All treated animal tissues, serum biochemical profiles, and total hemograms were normal. At 30-90 mg/kg oral doses, CAR-SNEDDS was not toxic and did not cause mortality. Conclusions: CAR-SNEDDS was successfully synthesized and characterized, and the results from sub-chronic oral toxicity studies showed that the CAR-SNEDDS were non-toxic and safe for biomedical fields.