Graphene oxide (GO) features distinctive physical and chemical characteristics; therefore, it has been intensively investigated in environmental remediation as a promising material for clean-up of soil contamination and water purification and used as immobilization material. Plastic is a widespread pollutant, and its breakdown products such as nanoplastics (NPs) should be evaluated for potential harmful effects. This study is aimed to evaluate the influence of GO on the toxicity of polystyrene (PS) NPs to the marine microalgae Picochlorum sp. over a period of 4 weeks. The capability of GO to reduce the toxic effects of PS NPs was assessed through investigating exposure sequence of GO in the presence of 20 nm diameter-sized polystyrene NPs. This was accomplished through five test groups: microalgae pre-exposed to GO prior to incubation with PS NPs, microalgae post-exposed to GO after incubation with PS NPs, microalgae simultaneously exposed to GO and PS NPs, and individual exposure of microalgae to either GO or PS NPs. Cytotoxicity assay results demonstrated that microalgae pre-exposed to GO prior to incubation with PS NPs showed an increased viability and chlorophyll a content. The pre-exposure to GO has reduced the growth inhibition rate (IR) from 50%, for microalgae simultaneously exposed to GO and PS NPs, to 26%, for microalgae pre-exposed to GO. Moreover, the lowest level of reactive oxygen species (ROS) was recorded for microalgae exposed to GO only and microalgae pre-exposed to GO. Fourier-transform infrared (FTIR) analysis, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) observations revealed some morphological changes of both algae and their extracellular polymeric substances (EPS) upon GO and PS NPs exposure combinations. The sequence of GO exposure to aquatic microorganisms might affect the level of harm caused by the PS NPs. Therefore, application of GO as part of an immobilization material and in the removal of pollutants from water should be carefully investigated using different pollutants and aquatic organisms.