This study investigates how variations in oxidation state and flake size influence the in vivo toxicity of graphene oxide (GO) using Caenorhabditis elegans as a model organism. Four GO types with distinct physicochemical properties were characterized and evaluated for effects on viability, locomotion, pharyngeal function, and mitochondrial ROS production. Highly oxidized materials, particularly smaller-flake GO4, exhibited greater acute and subacute toxicity and increased oxidative stress. Results demonstrate that both oxidation level and lateral size are critical determinants of biological impact, supporting grouping and read-across strategies for nanosafety and environmental risk assessment.