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Microorganisms frequently co‐exist in matrix‐embedded multispecies biofilms. Within biofilms, interspecies interactions influence the spatial organization of member species, which likely play an important role in shaping the development, structure and function of these communities. Here, a reproducible four‐species biofilm, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, was established to study the importance of individual species spatial organization during multispecies biofilm development. We found that the growth of species that are poor biofilm formers, M. oxydans and P. amylolyticus, were highly enhanced when residing in the four‐species biofilm. Interestingly, the presence of the low‐abundant M. oxydans (0.5% of biomass volume) was observed to trigger changes in the composition of the four‐species community. The other …