Multispecies biofilms are communities composed of different microorganisms embedded in an auto-synthesized polymeric matrix. This matrix allows potential inter-species interactions that play an important role in chronic infections. Pseudomonas aeruginosa and Burkholderia cenocepacia are two multidrug-resistant and biofilm-forming opportunistic pathogens often found in the lungs of cystic fibrosis patients. In this context, planktonic, static and dynamic biofilms and in vivo models of both species were optimized to understand the population dynamics and disposition, virulence and antibiotic susceptibility. From those models we determined that B. cenocepacia grows in a clustered manner at the bottom layers of biofilms, in close contact with P. aeruginosa, that tends to occupy the top layers. The coexistence of the studied bacteria resulted in a general increase of virulence-related gene expression in both species at early stages of coinfection and in in vivo models, while there was a general repression of virulence-related genes after longer coexistence periods. When evaluating antimicrobial susceptibility, a decrease of antimicrobial tolerance was observed in both species when in coculture. These findings shred a light on the differential behavior of P. aeruginosa and B. cenocepacia in dual-species systems, stressing the relevance of multispecies studies in the clinical context.