Display omitted •Biofilm formation rates were monitored using interfacial rheology.•Viscoelastic properties were correlated with key matrix components production.•Biofilm formation kinetics determined the final biofilm composition.•V. cholerae inhibited the production of curli unless E. coli was given a head-start. Biofilm is the dominant microbial form found in nature, in which bacterial species are embedded in a self-produced extracellular matrix (ECM). These complex microbial communities are responsible for several infections when they involve multispecies pathogenic bacteria. In previous studies, interfacial rheology proved to be a unique quantitative technique to follow in real-time the biofilm formation at the air-liquid interface. In this work, we studied a model system composed of two bacteria pathogenic capable of forming a pellicle biofilm, V. cholerae and E. coli. We used an integrated approach by combining a real-time quantitative analysis of the biofilm rheological properties, with the investigation of major matrix components and the pellicle microstructure. The results highlight the competition for the interface between the two species, driven by the biofilm formation growth rate. In the dual-species biofilm, the viscoelastic properties were dominated by V. cholera, which formed a mature biofilm 18 h faster than E. coli. The microstructure of the dual-species biofilm revealed a similar morphology to V. cholerae alone when both bacteria were initially added at the same amount. The analysis of some major ECM components showed that E. coli was not able to produce curli in the presence of V. cholerae, unless enough time was given for E. coli to colonize the air-liquid interface first. E. coli secreted phosphoethanolamine (pEtN) cellulose in the dual-species biofilm, but did not form a filamentous structure. Our pathogenic model system demonstrated the importance of the biofilm growth rate for multispecies biofilm composition at the air-liquid interface.