This study evaluates the bioavailability, developmental toxicity, genotoxicity, and estrogenic effects (EA) of resveratrol nanoparticles (NPs) stabilized by Tween 80 and carboxymethyl chitosan (CMCS) using chicken embryonic models. Tween NPs exhibited smaller sizes (93.18 nm), and more homogeneous distribution compared to CMCS NPs (267.92 nm). Resveratrol-loaded CMCS NPs achieved a peak serum concentration (Cmax) of 0.462 μg/mL at 60 minutes, while Tween NPs showed lower bioavailability (Cmax = 0.16 μg/mL). CMCS NPs induced a higher mortality (45.0%) at 760 μg/kg compared to Tween NPs (25.0%) and resveratrol (0%). Without loaded with resveratrol, Tween Empty NPs and CMCS Empty NPs showed higher mortality (51.5% and 60.7%, respectively). As capping agents, Tween 80 showed minimal mortality but induced malformation of 15.8% at higher dose. CMCS increased mortality (30%) and malformation (10%) at 10 mg/kg. Empty NPs induced higher lipoperoxidation and DNA damage than resveratrol-loaded NPs. Tween Empty NPs significantly elevated DNA damage (p <
0.0001), while CMCS Empty NPs exhibited no genotoxic effects. Estrogen-responsive apolipoprotein II (ApoII) gene expression was significantly upregulated in the Tween Empty group (9.51 ± 3.27-fold), indicating potential EA of the nanocarrier. These findings highlight both the NPs and nanocarriers must be evaluated in safety of nano-formulations.