Technological development using graphene oxide (GO) has increased in the last years, leading to the release of this contaminant to final sinks, such as estuaries. Due to their potential to flocculate and deposit when interacting with high ionic strength media, GO poses a threat, especially to benthic organisms like polychaetes. In addition to chemical contamination, estuaries also face a severe threat from invasive species, which can cause irreversible damage to ecosystems. The combination of abiotic and biotic stressors may work together on native species, decreasing their resilience. Thus, this study aims to assess the effects of an abiotic stressor, GO nanosheets (0.001, 0.01, 0.1, 1, 10mg GO/Kg dw) on Hediste diversicolor (native species) and Arenicola marina (invasive species) through several behavioral assays and biochemical markers. The impact of invasive species A. marina (biotic factor) on H. diversicolor avoidance behavior was also evaluated. Obtained results demonstrated that H. diversicolor fled from lower GO contamination compartments to higher ones and that exposure to increased GO concentrations negatively impacted its burrowing activity. They were unable to escape from higher contamination compartments, but at the highest concentrations, the bioturbation activity was significantly higher, which may indicate that H. diversicolor tended to dwell deeper in the sediment. A. marina showed an escape behavior from compartments with higher GO concentrations. Additionally, this species' bioturbation activity significantly decreased when exposed to GO. Moreover, avoidance tests demonstrated that the presence of A. marina affected the behavior of H. diversicolor. Regarding oxidative stress, H. diversicolor seems to be more impacted than A. marina, since Lipid peroxidation levels were higher in all GO concentrations and Superoxide dismutase activity significantly increased in the lowest GO levels. Overall, H. diversicolor spatial distribution may be severely constrained under abiotic and biotic stress, while A. marina's higher foraging activity may promote its propagation in the estuary. Behavioral tests, combined with biochemical markers have shown to be relevant tools for the development of more environmental-realistic assessment and monitoring frameworks for estuaries.