Actinobacillus pleuropneumoniae is the etiological agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Multispecies biofilms are probably the dominant form of microbial growth in nature and clinical data suggest that bacterial species form multi-species biofilms in vivo. The goal of this study was to determine the capacity of A. pleuropneumoniae to form multi-species biofilms with other swine pathogens (Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus and Escherichia coli). A serotype 1 field isolate of A. pleuropneumoniae, strain 719, was used in a 96-well microtiter plate biofilm assay. A. pleuropneumoniae was able to form strong two-species biofilms in the presence of S. suis, B. bronchiseptica and S. aureus under growth conditions that are not favorable for A. pleuropneumoniae (BHI media without NAD supplementation) when both species were added at the same time. When inoculated with P. multocida or E. coli, A. pleuropneumoniae biofilm formed a weak biofilm. In all cases, viable bacteria were recovered from the two-species biofilms. The live and dead populations, and the matrix composition of the multi-species biofilms were characterized using fluorescent markers (SYTO-9, propidium iodide, wheat germ agglutinin, Sypro Ruby and BOBO-3) and enzymatic treatments (dispersin B, proteinase K and DNase). The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure. A. pleuropneumoniae was also able to incorporate into pre-formed biofilms of S. aureus, S. suis or E. coli, under conditions that favour biofilm formation by S. aureus, S. suis or E. coli but not A. pleuropneumoniae. In conclusion, our data suggest that A. pleuropneumoniae is able to acquire pyridines compounds from other swine pathogens and form or incorporate into biofilms with other swine pathogens.