Industrial hemp is a multi-use crop that has been widely cultivated to produce fibers and nutrients, such as protein, dietary fiber, minerals, and unsaturated fatty acids, which make them a good fortifying component in food production. The antioxidant capability of hemp oils has been reported. In the current study, for evaluating the antioxidant activity of commercial hemp oil "Annabis BIO" derived from certified industrial hemp seeds without the psychoactive substance THC (Olomouc, Czech Republic), biomarkers of oxidative stress [2-thiobarbituric acid reactive substances (TBARS), oxidatively modified proteins (OMP), total antioxidant capacity (TAC)] were used in models of the blood collected from adult healthy rainbow trout (Oncorhynchus mykiss Walbaum), the blood of rainbow trout with clinical symptoms of ulcerative dermal necrosis (UDN), and equine plasma. A volume of 0.1 mL of the hemp oil was added to 1.9 mL of fish blood or equine plasma. After incubation of the mixture for 60 min with continuous stirring, biomarkers of oxidative stress were studied in samples. After in vitro incubation of hemp oil with the blood of clinically healthy rainbow trout, we noted a statistically significant decrease in biomarkers of lipid peroxidation by 55.6% (p <0.05). The highest increase in TBARS level was observed after in vitro incubation of hemp oil with the blood of UDN-affected rainbow trout. In vitro incubation of hemp oil with equine plasma resulted in a statistically significant increase in the level of ketonic derivatives (by 29%, p <0.05) and aldehydic derivatives of OMP (by 33.1%, p <0.05). Incubation of hemp oil with the blood of UDN-affected trout resulted in a decrease of the ketonic derivative of OMP (by 43.3%, p <0.05). Incubation of hemp oil with equine plasma, we observed a statistically significant decrease in TAC level by 56.6% (p <0.05). Similarly, after incubation hemp oil with blood samples of UDN-affected trout, a statistically significant decrease in total antioxidant capacity (by 59.3%, p <0.05) was observed. The results suggest that the investigated hemp oil have shown varied antioxidant capacities. Accordingly, this study proposes that the therapeutic benefit of this hemp oil can be, at least in part, attributed to using different biological materials (blood, plasma) used in vitro in the current study.