Diplodia seriata and Phaeomoniella chlamydospora are two fungal pathogens associated with grapevine trunk diseases worldwide. This study aimed to evaluate the period during which grapevine pruning wounds remain susceptible to fungal infection and to describe the colonization of canes artificially inoculated with these pathogens. In the first experiment, pruning wounds made in either fall or winter were separately inoculated with each pathogen at different times after pruning. Wound susceptibility to both pathogens decreased as the period between pruning and inoculation increased, from high percentages recorded in the first inoculation round (D. seriata, 97.5% and P. chlamydospora, 75%) down to approximately 10% 12 weeks after pruning. Pruning wounds remained more susceptible to D. seriata after a late pruning in winter whereas no overall seasonal changes in wound susceptibility were detected for P. chlamydospora. In the second experiment, canes were pruned by leaving two different lengths between the top node and the pruning wound before inoculations. Pathogens were recovered at different incubation periods and from different sites along the canes to estimate fungal cane colonization. A longer pruned internode made cane colonization by P. chlamydospora difficult, as indicated by fungal recoveries lower than 10% at the lowest recovery site, whereas D. seriata was less inhibited.