With the rapid development of nanotechnology, graphene oxide (GO) has increasingly been produced and widely applied in many fields, resulting in its environmental release and exposure to plants. Endophytic fungi can colonize the internal tissue of the plant without causing harm on their host, forming a mutually beneficial symbiotic relationship with the host plant. To date, the impacts of GO exposure on the fungal endophytic community in terrestrial plants have been rarely reported. In the present study, ryegrass (Lolium perenne L.) seeds were exposed to varying concentrations of GO solution (0.4%, 0.8%, and 1.2%) for a duration of 4 days to assess the impact of GO on the composition and diversity of endophytic fungi within ryegrass seeds. A high-throughput sequencing technique was utilized to analyze the alterations in the endophytic fungal community as affected by GO. Our results revealed the presence of 303 fungal species across all samples, categorized into 10 phyla, 39 classes, 84 orders, 160 families, and 240 genera. Predominantly, Ascomycota and Basidiomycota were identified as the dominant fungal phyla, with Pleosporales and Alternaria being the common dominant fungal order and genus, respectively. Importantly, the fungal endophytes within ryegrass seeds demonstrated sensitivity to GO exposure, particularly with the 1.2% GO treatment. As the concentration of GO increased, the abundance of Ascomycota exhibited a significant reduction of 19% and 20% for the 0.8% and 1.2% GO treatments, respectively, in comparison to the control (P<0.05). On the other hand, the abundance of Basidiomycota was significantly higher in all GO treatments compared to the control (P<0.05). Specifically, in the case of the 1.2% GO treatment, the abundance of Pleosporales and Alternaria notably decreased by 44.48% and 37.36%, respectively, compared to the control. Furthermore, exposure to GO altered the richness and diversity of the fungal endophytic communities in ryegrass seeds. The presence of GO at 1.2% resulted in significant increases (P<0.05) of 123.5%, 127.4%, and 117.5% in ACE, Chao1, and Shannon indices, respectively, relative to the control. Hierarchically clustered heatmap of fungal distribution at the genus level and Principal Coordinate Analysis (PCoA) highlighted significant disparities in the community structure of endophytic fungi between the 1.2% GO and the other three treatments. Additionally, LEfSe analysis revealed noteworthy distinctions in indicator taxa among the different treatments. Our findings underscore the influence of GO on altering the composition and diversity of endophytic fungal communities within ryegrass seeds, particularly evident in high-concentration treatments (1.2%). This study serves as a valuable reference for assessing the potential impact of carbon nanomaterial exposure on cohabiting species.