Nanotechnology has been extensively used in the development of drug delivery systems, with various benefits on the protection, control release, and improvement of drug delivery to target sites. Among nanocarriers, lipid-based ones, such as solid lipid nanoparticles (SLNs), have been biocompatible and theoretically safe for pharmaceutical application. Even so, studies evaluating the possible toxicity of nanocarriers have been dismissed and are lacking in the literature. Therefore, our study aimed to evaluate the toxicity of SLNs and their components using in vitro and in vivo studies. The in vitro studies were carried out in a normal human fibroblast cell line. The formulations of positively charged SLNs (SLNs⁺) exhibited greater cytotoxicity compared to negatively charged ones (SLNs^-), especially at higher concentrations (10, 20, and 100µg/mL), suggesting that this response is probably due to the higher interaction of SLNs⁺ with cell membranes. The SLN components (Precirol^® ATO 5, Tween^® 80, and benzalkonium chloride) were also studied in their higher concentration within the formulation. Benzalkonium chloride was the only component that exhibited toxicity in the in vitro studies. The in vivo assays were performed using Drosophila melanogaster with SLN⁺ at 100µg/mL and their components. The toxicity was studied taking into consideration chronic and periodic exposition, and the parameters accessed were egg number, hatched flies, and sex. Considering chronic exposure, no statistical differences were found considering these parameters. When F₁ flies, submitted to chronical exposure, were crossed with non-treated flies, the effect on gender was evident when the different components were tested. The discrepancy between the in vitro and in vivo result suggests that while SLNs demonstrate toxicity in cell cultures, their effects are less pronounced in whole organisms, likely due to biological factors such as metabolism and detoxification mechanisms, highlighting the need for comprehensive in vivo testing to assess the true impact of nanocarriers.