AbstractQuestionsNumerous studies on community assembly processes have been conducted in natural ecosystems. However, we know little about community assembly processes in human‐dominated urban ecosystems. Here, we asked: (1) how are the composition and functional diversity of native and exotic plant species shaped by local environment and landscape factors across urban vacant lots; and (2) how is microbial (bacterial and fungal) community composition influenced by the local environment, landscape factors, and plant species composition across urban vacant lots?LocationWe investigated 69 urban vacant lots in Yokohama, Japan.MethodsBy using a variation partitioning approach, we examined the relative importance of local environmental and landscape factors (including land use and spatial structure) in explaining variation in plant species composition and functional diversity of native or exotic species. We also explored the relative importance of local environmental and landscape factors, and plant species composition in explaining variation in microbial community composition.ResultsThe spatial structure of vacant lots determined the species composition and functional diversity of plant communities, suggesting that plant community assembly is determined by dispersal limitation. However, the functional diversity of the exotic species varied randomly, which reduced the relative importance of the spatial structure of vacant lots. Plant species composition as well as the spatial structure of vacant lots were the important drivers of the composition of soil microbial communities, despite a higher proportion of unexplained variation in their composition. Finally, we found an essential contribution of earthmoving methods in explaining the variations in both plant and microbial community composition.ConclusionPlant and microbial community composition would be largely determined by dispersal limitation across urban vacant lots. Understanding urban community assembly is critical for predicting plant and microbial communities that play an essential role in regulating urban ecosystem functioning and services.