Foliar endophytic fungi (FEF) are diverse and ubiquitously associated with photosynthetic land plants. However, processes shaping FEF assemblages remain poorly understood. Previous studies have indicated that host identity and host habitat are contributing factors, but these factors are often difficult to disentangle. In this study, we studied FEF assemblages from plants grown in a botanical garden, enabling us to minimize the variation in abiotic environmental conditions and fungal dispersal capacity. FEF assemblages from 46 Ficus species were sequenced using next-generation methods, and the results indicated that closely related host species had clearly differentiated FEF assemblages. Furthermore, host phylogenetic proximity was significantly correlated with the similarity of their FEF assemblages. In the canonical correspondence analysis, eleven leaf traits explained 32.9% of the total variation in FEF assemblages, whereas six traits (specific leaf area, leaf N content, leaf pH, toughness, latex alkaloid content, and latex volume per leaf area) were significant in the first two dimensions of ordination space. In the multiple regression on distance matrix analysis, 21.0% of the total variance in FEF assemblage was explained by both host phylogeny and leaf traits while phylogeny alone explained 7.9% of the variance. Thus, our findings suggest that both evolutionary and ecological processes are involved in shaping FEF assemblages.