Cannabis sativa L. with high cannabinoid content holds significant importance due to its diverse therapeutic and industrial applications. High cannabinoid varieties, such as those rich in CBD (cannabidiol) and low in THC (Δ⁹-tetrahydrocannabinolΔ⁹-Δ⁹-tetrahydrocannabinol), are highly sought after for their medicinal properties, including pain relief, anti-inflammatory effects, and potential benefits for mental health conditions like anxiety and depression. The selection and development of new hemp varieties with optimized cannabinoid profiles are crucial for enhancing the efficacy and specificity of cannabis-based treatments. This study evaluated 82 phenotypically different hemp selections resulting from the crossing of two hemp genotypes: the first being cv. Marina (from Serbia, included in the European List of Approved Hemp Varieties) and the second was a cannabinoid-type hemp selection from the United States. The CBD content in the drymaterial ranged from 0.207 % (in selection DZ 17) to 14.74 % (DZ 72), and the THC content ranged from 0.085 % (DZ 17) to 0.45 % (DZ 13). Additionally, 32 selections were chosen for evaluation of the essential oil (EO) content and composition. The major EO constituents were β-caryophyllene, α-humulene (α-caryophyllene), nerolidol, caryophyllenyl alcohol, and caryophyllene oxide. Additionally, CBD and traces of cannabichromene and dronabinol were detected in the EO. We identified 17 distinct chemovars demonstrating a significant variation in chemical profile. The EOs from novel hemp lines exhibited strong antimicrobial activity, particularly against Gram-negative bacteria and foodborne pathogens. DZ68 and DZ64 were the most effective, significantly inhibiting Escherichia coli, Salmonella enterica, and Listeria monocytogenes. These findings highlight the potential of these EOs for pharmaceutical and industrial applications, especially in natural antimicrobial formulations, and support the development of promising new hemp varieties.