Flavonols are dietary polyphenols whose biological activity is influenced by structural modifications such as O-methylation. This study compared two quercetin derivatives, isorhamnetin (3′-O-methylquercetin) and rhamnetin (7-O-methylquercetin). Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP) assays. Cyclooxygenase-2 (COX-2) inhibitory activity was assessed in vitro and supported by molecular docking simulations. In vivo effects included developmental toxicity, behavioral assessment, and locomotor responses in a 6-hydroxydopamine (6-OHDA) model. The results demonstrated that rhamnetin exhibited significantly stronger radical-scavenging and reducing activity in DPPH, ABTS, and FRAP assays, whereas no significant differences were observed in the CUPRAC assay. Isorhamnetin showed stronger COX-2 inhibition, with docking results suggesting a different mode of binding when analyzing possible interactions with enzyme active site. In zebrafish larvae, rhamnetin showed lower observable developmental toxicity within the tested concentration range, whereas isorhamnetin induced developmental abnormalities at higher concentrations. Both flavonols attenuated 6-OHDA-associated locomotor deficits and modulated antioxidant enzyme activity under oxidative stress conditions. In conclusion, our findings indicate that the position of O-methylation influences flavonol antioxidant properties, COX-2 interactions, and organism-level responses.