Background. Blackthorn (Prunus spinosa L.) fruits are rich in bioactive compounds, including tannins, flavonoids, and anthocyanins, known for their antioxidant properties and potential applications in functional foods. Goals. This study investigated the effects of harvest timing in summer and winter and in solvents such as distilled water and 70% ethanol on the chemical composition and antioxidant activity of blackthorn fruit extracts. Methodology. Following analytical protocols, the total phenols and tannins, flavonoids, and anthocyanins were determined spectrophotometrically. The tannin content was determined by the difference between total phenols and non-tannic phenols. The efficiency of natural antioxidants in the tested samples was determined spectrophotometrically using the FRAP, ABTS+, and DPPH methods. Results. The results reveal significant seasonal and solvent-based differences in the chemical composition and antioxidant properties of blackthorn fruits. Winter-harvested fruits consistently show higher concentrations of bioactive compounds compared to summer fruits. Ethanol consistently proves to be a more efficient solvent, yielding higher values for all measured parameters across both seasons, although the seasonal differences are less pronounced in ethanol extracts. Fruits collected during winter, with their higher antioxidant capacity and bioactive content, are particularly promising for applications in functional foods or nutraceuticals, especially when processed using ethanol-based extraction methods. Ethanol consistently outperformed water as a solvent, yielding higher concentrations of bioactive compounds and antioxidant activity. Winter-harvested fruits showed a significant increase in tannins (104% higher in water extracts and 50% in ethanol extracts), flavonoids (93% higher in water extracts), and anthocyanins (61% higher in water extracts), while summer ethanol extracts had 15% more anthocyanins. Antioxidant activity, assessed through FRAP, DPPH, and ABTS assays, was notably greater in autumn fruits, with water extracts showing 35–49% higher activity and ethanol extracts displaying 9.5–24% higher activity. Conclusions. These results highlight the critical role of harvest timing and solvent choice in maximizing the extraction of bioactive compounds and antioxidant potential. Winter fruits, especially when extracted with ethanol, are particularly valuable for developing health-promoting products, emphasizing the importance of optimizing these factors to enhance the nutritional and functional properties of blackthorn fruits.