Human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor within the ErbB family that plays a pivotal role in the progression of various aggressive cancers. HER2-positive tumors often develop resistance to standard therapies, necessitating the exploration of innovative treatment options. Cannabinoids, bioactive compounds from such as cannabidiol (CBD), cannabigerol (CBG), and cannabinol (CBN), have gained attention for their potential anticancer properties. This study evaluates the efficacy of CBD, CBG, and CBN in targeting HER2-positive ovarian cancer through kinase inhibition assays, surface plasmon resonance (SPR), molecular docking, and cell viability assessments. SPR analysis revealed that cannabinoids bind strongly to HER2-tyrosine kinase (HER2-TK), with CBD showing the highest affinity ( = 6.16 μM), significantly better than afatinib ( = 26.30 μM), and CBG demonstrating moderate affinity ( = 17.07 μM). In kinase inhibition assays, CBG was the most potent inhibitor (IC = 24.7 nM), followed by CBD (IC = 38 nM), suggesting their ability to disrupt HER2-mediated signaling pathways. Molecular docking studies highlighted critical interactions between cannabinoids and essential HER2 residues (Leu796, Thr862, Asp863). In cell viability assays, CBD and CBG effectively inhibited the growth of HER2-positive SKOV3 cells (IC = 13.8 μM and 16.6 μM, respectively), comparable to traditional tyrosine kinase inhibitors. These findings underscore the therapeutic potential of cannabinoids, particularly CBD and CBG, as alternative or adjunct therapies for HER2-positive cancers, with the promise of mitigating resistance and adverse effects associated with existing treatments.