Abstract Environmental microbes frequently live in multispecies biofilms where mutualistic relationships and co-evolution may occur, defining spatial organization for member species and overall community functions. In this context, intrinsic properties emerging from microbial interactions, such as efficient organization optimizing growth and activities in multispecies biofilms, may become the object of fitness selection. However, little is known on the nature of underlying interspecies interactions during establishment of a predictable spatial organization within multispecies biofilms. We present a comparative metatranscriptomic analysis of bacterial strains residing in triple-species and four-species biofilms, aiming at deciphering molecular mechanisms underpinning bacterial interactions responsible of the remarkably enhanced biomass production and associated typical spatial organization they display. Metatranscriptomic profiles concurred with changes in micro-site occupation in response to the addition/removal of a single species, being driven by both cooperation, competition, and facilitation processes. We conclude that the enhanced biomass production of the four-species biofilm is an intrinsic community property emerging from finely tuned space optimization achieved through concerted antagonistic and mutualistic interactions, where each species occupies a defined micro-site favoring its own growth. Our results further illustrate how molecular mechanisms can be better interpreted when supported by visual imaging of actual microscopic spatial organization, and we propose phenotypic adaptation selected by social interactions as molecular mechanisms stabilizing microbial communities.