Plant-derived extracellular vesicles (EVs) have been the topic of interest in recent years due to their proven therapeutic properties. Intact or manipulated plant EVs have shown antioxidant, anti-inflammatory, and anti-cancerous activities as a result of containing bioactive metabolites and other endogenous molecules. Less is known about the EV efficacy with high levels of bioactive secondary metabolites derived from medicinal or non-edible plants. Numerous data suggest the functionality of Cannabis sativa extract and its phytocannabinoids in cancer treatment. Here, two chemotypes of cannabis with different levels of D-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) were selected. EVs were isolated from each chemotype via differential ultracentrifugation. HPLC analysis was illustrative of the absence of THC in EVs derived from both plants. Therefore, two types of EVs were classified according to their CBD content into high- (H.C-EVs) and low-CBD EVs (L.C-EVs). Electron microscopy and DLS showed both cannabis-derived EVs (CDEVs) can be considered as exosome-like nanovesicles. Cytotoxicity assay showed that H.C-EVs strongly decreased the viability of two hepatocellular carcinoma (HCC) cell lines, HepG2 and Huh-7, in a dose and time-dependent manner compared with L.C-EVs. H.C-EVs had no significant effect on HUVECs normal cell growth. The finding showed that the H.C-EVs arrested the G0/G1 phase in the cell cycle and significantly induced cell death by activating mitochondrial-dependent apoptosis signaling pathways in both HCC cell lines. Altogether, the current study highlights that CDEVs can be an ideal natural vehicle for bioactive phytocannabinoids and a promising strategy in cancer management.