The aim of this study was to compare the micro‐morphological features of two different non‐drug Cannabis sativa L. biotypes (Chinese accession G‐309 and one fibrante variety) and to evaluate the phytochemical profile as well as some biological properties of the essential oils (EOs) obtained by hydrodistillation of dried flowering tops. After a micro‐morphological evaluation by scanning electron microscopy, the phytochemical composition was analysed by GC–FID and GC–MS analyses. Antioxidant and anti‐acetylcholinesterase properties were investigated by several in vitro cell‐free assays, while neuroactive effects were evaluated on mouse cortical neuronal as well as human iPS cell‐derived central nervous system cells grown on MEA chips. Both EOs showed strong antioxidant properties mainly attributable to the high content of hydroxylated compounds as well as significant anti‐acetylcholinesterase activities (IC50 74.64 and 57.31 μg/ml for Chinese accession and fibrante variety, respectively). Furthermore, they showed a concentration‐dependent inhibition of spontaneous electrical activity of human and mouse neuronal networks, with the fibrante variety, which showed the best activity (MFR, IC50 0.71 and 10.60 μg/ml, respectively). The observed biological activities could be due to a synergic effect between terpenes and phytocannabinoids, although in vivo studies, which clarify the molecular mechanism, are still lacking.