The paper presents the overall results and experimental details of the in vitro assessment of the elderberry (Sambucus nigra) extract on the motility, viability and reactive oxygen species (ROS) production of bovine spermatozoa in different time periods (0, 2, 6 and 24 hours). Sambucus nigra is often used for medicinal purposes throughout the world. Modern research reveals that Sambucus nigra extracts may have anti-inflammatory, antiviral, anticancer and antioxidant properties, because of a high content of biologically active components. Spermatozoa motility was assessed using the Computer-assisted sperm analysis (CASA) system. Cell viability was examined using the metabolic activity MTT assay and ROS generation was quantified using luminometry. The CASA analysis revealed that the motility in the experimental groups supplemented with 100, 50 and 1 μg/mL elderberry extract was lower in comparison with other samples. The experiment showed that the elderberry extract had a considerable in vitro effect on the sperm motility, vitality and oxidative profile. The ROS production as well as the CASA assessment proved that the optimal concentration of both extracts was 10 μg/mL in every time with statistically significant results. The MTT test showed a statistically significant increase of mitochondrial at all time periods with 10 μg/mL elderberry extract when compared to the control group. When lower concentrations of the elderberry extract were used (5 and 1 μg/mL), the mitochondrial activity was higher than in the control group but lower than in the group supplemented with 10 μg/mL of the extract. In these groups this indicator increased maximally after 24 h. The findings of the present study indicate that Sambucus nigra extract possesses activity promoting properties on bovine spermatozoa at 10 and 5 μg/mL.