In this study, both chemical characterization and biological activities of propolis samples obtained from Cannabis sativa L. (cannabis, hemp, marijuana) isolated colonies (research group) and obtained from colonies at a distance where the bee cannot fly to the isolation area (control group) were determined. Palynological analyses of propolis samples obtained from the research and control group colonies were made and the botanical origin of the cannabis plant and other plants in the research area and the plants in the control group were determined. Bioactive component analysis of propolis extracts was determined by the GC–MS technique. The inhibition effect of propolis samples on some enzymes of medical importance and their antimicrobial effects against many pathogenic microorganisms were also investigated. Palynological analyses showed that honey bees prefer cannabis plants (11.11%) when collecting propolis, and the dry matter percentage (94.6 ± 1.8%), balsam (46.2 ± 5.2%), and wax content (4.8 ± 0.5%), total phenolic (26.44 ± 1.08 mg GAE/mL) and total flavonoid (2.03 ± 0.11 mg QE/mL) content and antioxidant capacity (88.16 ± 2.27 μM FeSO4·7H2O/mL−1) of the research group propolis samples containing hemp were found to be higher than the control group. Also, it was determined that the research group propolis sample contains cannabinoid derivatives (cannabidivarinic acid, 0.29%; delta (1)-tetrahydrocannabinolic acid, 0.59%; cannabidiolic acid, 0.48%), which are active compounds of the cannabis plant. Also, a high inhibition effect was detected on some enzymes of medical importance in propolis samples obtained from cannabis group colonies. In addition, the research group propolis samples also showed a high antimicrobial effect against many pathogenic microorganisms.