Nanoemulsions are dispersions of oil-in-water (O/W) and water-in-oil (W/O) immiscible liquids. Thus, our main goal was to formulate a nanoemulsion with low surfactant concentrations and outstanding stability using Copaiba balsam oil (Copaifera sp.). The high-energy cavitation homogenization with low Tween 80 levels was employed. Then, electrophoretic and physical mobility properties were assessed, in addition to a one- and two-year physicochemical characterization studies assessment. Copaiba balsam oil and nanoemulsions obtained caryophyllene as a major constituent. The nanoemulsions stored at 4 ± 2 °C exhibited better physical stability. Two years after formulation, the nanoemulsion showed a reduction in the particle size. The size underwent changes in gastric, intestinal, and blood pH, and the PdI was not changed. In FTIR, characteristic bands of sesquiterpenes and overlapping bands were detected. When subjected to freezing and heating cycles, nanoemulsions did not show macroscopic changes in higher concentrations. Nanoemulsions subjected to centrifuge force by 1000 rpm do not show macroscopic instability and phase inversion or destabilization characteristics when diluted. Therefore, the nanoemulsion showed stability for long-term storage. The nematode Caenorhabditis elegans was used to assess the potential toxicity of nanoemulsions. The nanoemulsion did not cause toxicity in the animal model, except in the highest concentration tested, which decreased the defecation cycle interval and body length. The toxicity and stability outcomes reinforce the nanoemulsions' potential for future studies to explore pharmacological mechanisms in superior experimental designs.