Purpose <p>Microglial activation is a key driver of neuroinflammation, contributing to the onset and progression of different neurodegenerative diseases. Essential oils (EOs) from <i>Cannabis sativa</i>, due to their complex phytochemistry, may represent a potential strategy to counterbalance neuroinflammation. Herein, the anti-inflammatory effects of two different EOs from agricultural waste of hemp genotypes, Eletta Campana and S435, were evaluated on a human model of microglial cells (HMC3).</p> Methods <p>The EOs were obtained by hydrodistillation with a Clevenger apparatus and analysed for their chemical composition by Gas-Chromatography coupled with Mass Spectrometry. To evaluate the biological activity of EOs, viability assay and radical oxygen species production (ROS) were performed. The EO anti-inflammatory activity was evaluated in terms of morphology, gene expression, and protein expression.</p> Results <p>EOs shared a cannabidiol-rich profile but differed in their sesquiterpene composition: Eletta Campana EO contained higher levels of β-caryophyllene and α-humulene. Conversely, S435 was rich in caryophyllene oxide. At non-cytotoxic concentrations, neither EOs nor their major compounds promoted the increase of intracellular ROS. Upon IL-1β stimulation, EO treatment attenuated microglial activation and significantly reduced the expression of pro-inflammatory genes IL-6, IL-8, and NF-κB. The EO inhibition of NF-κB nuclear translocation was confirmed with immunofluorescence analysis. As a possible mechanism of NF-κB inhibition, the reduction of ERK1/2 phosphorylation was demonstrated.</p> Conclusion <p>These findings suggest that <i>C. sativa</i> EOs can counteract microglial activation through the interaction of multiple bioactive compounds. The anti-inflammatory potential of Eletta Campana and S435 EOs derived from hemp supply chain by-products supports their value as promising candidates for neuroinflammation management in different neurodegenerative diseases.</p> Graphical Abstract <p></p>