The soil bacterium Bacillus subtilis forms beneficial biofilms that induce plant defences and prevent the growth of pathogens. It is naturally found in the rhizosphere, where microorganisms coexist in an extremely competitive environment, and thus have evolved a diverse arsenal of defence mechanisms. In this work, we found that volatile compounds produced by B. subtilis biofilms inhibited the development of competing biofilm colonies, by reducing extracellular matrix gene expression, both within and across species. This effect was dose-dependent, with the structural defects becoming more pronounced as the number of volatile-producing colonies increased. This inhibition was mostly mediated by organic volatiles, and we identified the active molecules as 3-methyl-1-butanol and 1-butanol. Similar results were obtained with biofilms formed by phylogenetically distinct bacterium sharing the same niche, Escherichia coli, which produced the biofilm-inhibiting 3-methyl-1-butanol and 2-nonanon. The ability of established biofilms to inhibit the development and spreading of new biofilms from afar might be a general mechanism utilized by bacterial biofilms to protect an occupied niche from the invasion of competing bacteria.