Willow (Salix spp.), a major source of biomass and renewable fiber production, is one of the best choices for short-rotation intensive culture (SRIC) in Canada. Since fungal communities play important roles in the plant’s health status, it is vital to understand their interactions with willows and their roles in the sustainability of SRIC. In this study, fungal diversity of the above-ground organs (stem/leaf) of healthy and diseased willow plants in western Canadian Prairies were assessed using cultural and PCR-denaturing gradient gel electrophoresis (DGGE) techniques. Comparison of the mycoprofiles within established plantations vs. newly introduced cuttings revealed differences in the fungal communities. Ascomycota were mainly isolated, followed by Basidiomicota and Zygomycota. Willow genotypes seem have an influence on the abundance of fungal pathogens and disease severity; among them Charlie (Salix alba x gladfelteri) and SV1 (S. eriocephala) cultivars demonstrated superior performances. Photosynthesis measurements and biomass compositions confirmed these findings. Potentially pathogenic fungi (Dothioraceae, Diaporthaceae, Glomeraceae, and Pleosporaceae) dominated in diseased or symptomatic willows, whereas potentially beneficial fungi (Coniochaetaceae, Hypoceraceae, Nectriaceae, Trichocomaceae, and Agaricaceae) prevailed in healthy plants. In-vivo and greenhouse assays showed that inoculation with potentially pathogenic fungi induced leaf necrosis, anthracnose and open cankers. However, suppression of the latter was still possible using fungal antagonists. Hence, assessment of stem/bark and leaf fungal communities with respect to willow genotypes, cuttings origin, and SRIC location, is useful for the design of an effective management strategy to increase the productivity of the SRIC-biomass systems.