Objectives The present study focused on exploring the chemical composition of essential oil and corresponding hydrolate obtained by steam distillation of industrial hemp ( Cannabis sativa L.) cultivar “Helena” (low THC content). Methods Chemical characterization of industrial hemp essential oil and hydrolate was performed by gas chromatographic and gas chromatographic-mass spectrometric analysis, while biological activities included antimicrobial and antioxidant tests. Antimicrobial activity was determined by measuring diameters of the inhibition zone by using a disc-diffusion method with nine microbial strains from ATCC culture. Moreover, minimal inhibitory concentration (MIC) as well as time-kill kinetic studies, antiadhesion, and antibiofilm formation potential were also evaluated. Antioxidant activity was evaluated through three different antioxidant assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH●), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS●+), and reducing power (RP). Results The gas chromatography mass spectrometry analysis showed that the main compound in the essential oil was trans-caryophyllene (37.4%), followed by caryophyllene oxide (12.4%) and α-humulene (11.0%), while in the hydrolate it was 1,8-cineole (11.5%). Results showed that industrial hemp essential oil and hydrolate exhibited no antimicrobial activity against gram-negative bacteria, yeasts, and fungi, while gram-positive bacteria were sensitive. Therefore, in the following step, MIC was determined by microdilution method. The lowest MIC for essential oil (12.5%) was obtained for Enterococcus faecalis and Staphylococcus aureus, while this value doubled for Listeria monocytogenes. Conversely, the MIC for hydrolate was 100% for all gram-positive bacteria. Antioxidant activity showed that industrial hemp essential oil and hydrolate have potential as natural sources of antioxidants. Conclusion This research confirmed the previously proven antimicrobial and antioxidant activities of industrial hemp essential oil. The novelty lies in the antimicrobial and antioxidant activity of hydrolate, which is practically waste, but has great potential to be a useful by-product.