The most important disease of European chestnut (Castanea sativa Mill.) is chestnut blight caused by the fungus Cryphonectria parasitica (Murrill) Barr which induces yield reduction in Europe and North America. This study aimed to investigate the impacts of C. parasitica infection on the physiological and biochemical characteristics of European chestnut at two different growth stages, 3 and 6 weeks after the infection. The amount of photosynthetic pigments (chlorophyll-a, chlorophyll-b, and carotenoids), the relative chlorophyll content, and the photochemical efficiency of the photosystem II (PSII) were measured in the leaves above and below the virulent and hypovirulent C. parasitica infections. The highest values were measured in the control leaves, the lowest values were in the leaves of the upper part of virulent necrosis. Antioxidant enzyme activities such as ascorbate peroxidase (APX), guaiacol peroxidase (POD), and superoxide dismutase (SOD), proline, and malondialdehyde concentrations were also investigated. In each of these measured values, the lowest level was measured in the control leaves, while the highest was in leaves infected with the virulent fungal strain. By measuring all of these stress indicator parameters the responses of chestnut to C. parasitica infection can be monitored and determined. The results of this study showed that the virulent strain caused more pronounced defense responses of chestnut’s defense system. The measured parameter above the infection was more exposed to the blight fungus disease relative to the leaves below the infection.