Biofilms that form on roots, litter and soil particles typically contain multiple bacterial species. Currently, little is known about multispecies biofilm interactions and few studies have been based on environmental isolates. Here, the prevalence of synergistic effects in biofilm formation among seven different soil isolates, cocultured in combinations of four species, was investigated. We observed greater biofilm biomass production in 63% of the four-species culture combinations tested than in biofilm formed by single-species cultures, demonstrating a high prevalence of synergism in multispecies biofilm formation. One four-species consortium, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, exhibited strong synergy in biofilm formation and was selected for further study. Of the four strains, X. retroflexus was the only one capable of forming abundant biofilm in isolation, under the in vitro conditions investigated. In accordance, strain-specific quantitative PCR revealed that X. retroflexus was predominant within the four-species consortium (>97% of total biofilm cell number). Despite low relative abundance of all the remaining strains, all were indispensable for the strong synergistic effect to occur within the four-species biofilm. Moreover, absolute individual strain cell numbers were significantly enhanced when compared with those of single-species biofilms, indicating that all the individual strains benefit from inclusion in the multispecies community. Our results show a high prevalence of synergy in biofilm formation in multispecies consortia isolated from a natural bacterial habitat and suggest that interspecific cooperation occurs.