Macrofungi are key functional components of forest ecosystems, contributing to nutrient cycling, organic matter decomposition and ecosystem stability. This study assessed macrofungal diversity and ecological distribution patterns in coniferous forests of “Bjeshkët e Nemuna” National Park (western Kosovo) during autumn 2025. Systematic sampling along altitudinal gradients was conducted across three localities using standardized 10 × 10 m plots. All visible fruiting bodies were recorded and identified through morphological and microscopic analyses. A total of 146 specimens were documented, including 108 species newly recorded for Kosovo, significantly expanding the national mycobiota. Functional analysis showed ectomycorrhizal fungi as the dominant ecological group, followed by terricolous and lichenicolous taxa, highlighting their essential role in forest nutrient dynamics and soil processes. Diversity indices indicated high species diversity and low dominance (Shannon H′ = 3.50–3.99; Simpson’s 1 − D > 0.96), while Jaccard similarity (0.293–0.412) demonstrated moderate spatial turnover. Species richness decreased with increasing elevation, suggesting that microclimatic variability and habitat heterogeneity strongly influence fungal distribution patterns. The findings provide baseline data for environmental monitoring and support the integration of macrofungi as bioindicators in sustainable forest management and conservation planning within mountainous ecosystems.