Nanotechnology is currently undergoing rapid development partly due to the increasing use of carbon-based nanoparticles, such as Few Layer Graphene (FLG). Owing to its numerous applications, its industrial production is likely to lead to environmental release, including into aquatic ecosystems. In this study, a transcriptomic approach was used to assess the effect of FLG at low (0.1 mg L-1) and high (50 mg L-1) concentrations on the benthic freshwater diatom Nitzschia palea after 48 h of exposure. Direct contact with FLG and induced shading were distinguished to compare the transcriptomic responses. Genes that were differentially expressed after exposure compared with control conditions were identified, and their functional descriptions are discussed. A slight transcriptomic response related to cell wall repair was observed in diatoms exposed to the low FLG concentration. Exposure to the high FLG concentration induced a strong response involving 1907 transcripts. Notably, 16 transcripts involved in the chlorophyll biosynthesis process were under-expressed (log2 fold change between -3 and -1.2), suggesting a down-regulation of the photosynthetic metabolism. Diatoms exposed to the high FLG concentration over-expressed about 13 transcripts encoding for extracellular proteins that play a role in cellular adhesion, and two transcripts involved in cell wall repair were highly up-regulated. Light deprivation caused by shading induced a downregulation of genes involved in the energetic metabolism of N. palea. Overall, these results revealed that metabolic pathways impacted by FLG exposure are concentration and contact dependent. Moreover, this study suggests that a low FLG concentration, close to that in environmental conditions, will have a minor impact on diatom biofilms whereas a high FLG concentration, mimicking accidental release, might be critical for ecosystems.