Nanoemulsions, characterized by their nanosized particles ranging from 20 to 200 nm, are effective carriers for drug molecules. Our novel oil-in-water nanoemulsion, NE-FLO™, formulated from lemon and fish byproduct oils, demonstrates promising antioxidant and anti-inflammatory activities, with initial studies indicating nontoxicity to normal skin cells. This study investigated the safety of NE-FLO™ using brine shrimp (Artemia salina) and zebrafish (Danio rerio) models, focusing on concentration-dependent effects and LC₅₀ values. At lower concentrations (0.1 mg·L⁻¹, 0.01 mg·L⁻¹, and 0.001 mg·L⁻¹), NE-FLO™ showed minimal toxicity without adverse effects. However, at 1 mg·L⁻¹, reduced survival rates indicate potential toxicity. Specifically, this concentration also induces altered swimming behaviors in zebrafish. LC₅₀ values are 8.7474 mg·L⁻¹ for brine shrimp and 0.316 mg·L⁻¹ for adult zebrafish. These results underscore the necessity for further detailed investigations into NE-FLO™, balancing its therapeutic benefits with potential toxicity risks. This study emphasizes the importance of optimizing nanoemulsion formulations from fish oil and conducting comprehensive safety assessments to meet regulatory standards.