ABSTRACT Grapevine pruning wounds made during the dormant season are a port of entry of wood-invading fungi. Timing of pruning may affect the wound susceptibility to these fungi, such as those associated with grapevine trunk diseases (GTDs). This study aimed to determine the effect of pruning time on natural fungal infection in six vineyards in Galicia, Spain, belonging to three Denominations of Origin (D.O) over two growing seasons. Pruning wounds were left unprotected physically and chemically during two periods of three months each, from November to February and from February to May. The diversity and composition of the fungal microbiome that colonized the pruning wounds was identified by ITS2 high-throughput amplicon sequencing (HTAS). A broad range of fungi was able to colonize grapevine pruning wounds at both infection periods. Fungal microbiome composition did not shift as year of sampling. Fungal communities were affected in their diversity and composition by the D.O., whereas the spatial variation (i.e. vineyard within each region) was low. Pruned canes harboured a core community of fungal species, which appeared to be independent of the infection period. Accumulated rainfall over 8 and 11 weeks after pruning positively correlated with the total fungal microbiome and in particular with the GTD fungal genus Diaporthe abundances. A strong seasonal effect on GTD fungal infection was detected for most genera, with higher percentages of abundance detected after pruning in February (winter) as compared with that of pruning in November (mid-autumn). In light of the GTD colonization results and given the environmental conditions and the geographical location of this study, early pruning is recommended to reduce the infections caused by GTD fungi during the pruning season in Galicia.