Biological invasions pose a major threat to biodiversity conservation in protected areas, with roads, tracks, and trails being the main pathways for the spread of non-native species. This study aimed to assess the distribution patterns of non-native and native plant species in relation to elevational gradient, public use intensity, and disturbance by roads and trails in a protected tropical mountain forest in southeastern Brazil. Specifically, we recorded plant species along this gradient and tested whether the richness of native and non-native species differed with elevation. Additionally, we investigated whether the high-altitude non-native species community was a subset of lower-elevation communities and whether non-native species richness was linked to anthropogenic disturbances and public use intensity. Our findings revealed that native and non-native species richness varied along the elevational gradient. Native species exhibited a hump-shaped pattern, with richness peaking at mid-elevations. In contrast, non-native species did not show a clear trend along the altitudinal gradient. Notably, higher non-native species richness was observed in roadside and trailside plots. The non-native species communities at higher altitudes were not simply subsets of those found at lower elevations. Thus, while the richness and composition of native species appeared to be driven by environmental factors along the elevational gradient, the presence of non-native species was more closely associated with anthropogenic disturbances. In summary, our results indicate that non-native plants, although widespread along trails and roads, establish primarily in the most disturbed areas. Therefore, roads, trails, and human and vehicular traffic are key determinants of biological invasions in this mountainous protected area.