Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation of E. coli cells in static cultures. Here, we show that Ag43 can be expressed in a functional form on the surface of Pseudomonas fluorescens. Ag43 expression dramatically enhances the biofilm-forming potential of both E. coli and P. fluorescens to abiotic surfaces in simple microtitre well assays and in flow chambers. Importantly, Ag43-expressing E. coli and P. fluorescens cells tagged with Gfp and Rfp were shown to form interwoven biofilms in flow chambers. The three-dimensional structures of the biofilms were analysed by laser-confocal microscopy. Heterogeneous expression of Ag43 induced interspecies cell-to-cell contact that generated multispecies biofilm formation. Our data indicate that this versatile molecular tool can be used for the rational design of multispecies biofilms. More specifically, this novel technology offers opportunities for the design of multispecies consortia in which the concerted action of several bacterial species is required, e.g. waste treatment and degradation of pollutants.