Branch canker and dieback, caused by Botryosphaeriaceae and Diaporthaceae, is a major disease of walnut (Juglans regia L.) worldwide. In Chile, the impact of pruning wound age and timing on susceptibility to these pathogens in walnut trees remains poorly understood. During June–July (2023) and June–July (2024), this study assessed the effect of pruning wound age of the walnut cv. Chandler on infection by seven fungal species and simultaneously tracked seasonal conidial release of Botryosphaeriaceae spp. in the Maule Region, Chile. Lignified twigs were artificially inoculated at 1, 15, 30, and 45 days after pruning, and necrotic lesion lengths were measured six months post-inoculation. All fungal isolates caused significantly longer lesions than the control (p < 0.0001), with Diplodia mutila, Neofusicoccum nonquaesitum, and N. parvum being the most aggressive. At the same time, Dothiorella sarmentorum and Diaporthe species (Diaporthe australafricana, Di. foeniculina, and Di. patagonica) produced the smallest lesions. Susceptibility decreased with increasing wound age, with a significant interaction between fungal species and pruning wound age. Spore trapping of Botryosphaeriaceae revealed that dispersal was positively associated with rainfall (r = 0.81, p < 0.0001), relative humidity (r = 0.51 to 0.61, p < 0.05) and average temperature (r = 0.32 to 0.58, p < 0.05), but negatively or not significantly correlated with maximum temperature (r = −0.59 to −0.79, p > 0.05). These results demonstrate that rainfall or relative humidity, moderate conditions, and favor conidial release. At the same time, infection risk declines with wound age, underscoring the need to adjust pruning schedules and preventive strategies to reduce disease risk in walnut orchards.