Mycoplasma hyopneumoniae (Mhp) is a chronic respiratory pathogen that causes significant losses to swine production worldwide. Disease involves colonisation of the respiratory tract cilia, causing ciliostasis and epithelial cell death. An understanding of the molecular mechanisms that underpin Mhp virulence is needed to develop efficacious vaccines. We show for the first time that Mhp can form biofilms on abiotic and biotic surfaces. This alternate lifestyle involves significant changes in protein expression and provides insight into new pathways to control this economically devastating pathogen. Time-lapse microscopy showed that prolific biofilms formed on glass after prolonged incubation (> 10 days) but formed in only 6 days on porcine epithelial cell monolayers (PK-15). We used a systems approach to identify Mhp proteins that bind PK-15 receptors. PK-15 surface proteins were biotinylated and bound to an avidin matrix and incubated with a native whole cell Mhp lysate. After extensive washing to remove unbound proteins, Mhp proteins bound to PK-15 cell receptor proteins were eluted in high salt followed by low pH to remove any strongly bound proteins. Eluted proteins were separated by SDS-PAGE, in-gel digested with trypsin and analysed by LC-MS/MS. Notably, ~75% of the Mhp proteins that bound to PK-15 cell receptors were previously identified to reside on the cell surface of Mhp. One of these was Mhp271, a protein previously shown to be a cilium adhesin; validating our methodology. Notably, Himar mutants located in Mhp271 are also unable to form biofilms. We also identified a number of additional putative biofilm associated genes using a high-throughput biofilm assay developed in our lab. Further work is underway to characterise these proteins and examine their role in the pathogenesis of Mhp.