The presence of Cryphonectria parasitica (Murrill) M.E. Barr was studied in six natural and planted stands of sweet chestnut in Serbia. The fungus was detected on the sweet chestnut in five localities and on the sessile oak in one locality. In total, 77 isolates from the sweet chestnut and five isolates from the sessile oak were obtained. Based on the culture morphology, all the obtained isolates were proven to be free from Cryphonectria (Saccardo) Saccardo & D. Saccardo hypovirus. The isolates of C. parasitica from the sweet chestnut were compatible with three different vegetative compatibility types, EU-12, EU-2, and EU-1, while the isolates from the sessile oak belonged to EU-12. After inoculation in laboratory conditions, the isolate from the sweet chestnut and sessile oak caused the decline in 88 and 76% of the sweet chestnut plants, respectively. In the case of the sessile oak, both isolates caused the decline in 52% of the plants. In field conditions, both isolates were aggressive to sessile oak trees after previous bark wounds and they were statistically significantly different compared to the control trees. The isolate from the sweet chestnut caused significantly larger cankers compared to both the isolate from the sessile oak and the control.