Evidence of biofilms in human chronic wounds are thought to be responsible for preventing healing in a timely manner. However, biofilm evidence in horse wounds has not yet been documented. Consequently, this study aimed to determine whether biofilms could be detected in wounds, and to investigate the microbiology of chronic wounds in horses. Prior to analysis, wound surfaces were irrigated with 5 mL of sterile saline to remove debris. All wounds were swabbed twice (1 cm2 area) using sterile cotton-tipped swabs. In addition to this, 2 tissue biopsies were taken to investigate evidence of biofilm and the microbiology richness of the wounds. All swabs and 1 biopsy sample were transported to the laboratory in Robertson's cooked meat broth. Traditional culturable techniques and denaturing gradient gel electrophoresis with PCR were utilized to identify common bacteria isolated in all wounds. Following analysis of a number of the biopsy samples, biofilms could be clearly seen. The most common bacteria isolated from each wound analysed included Pseudomonas aeruginosa, Staphylococcus epidermidis, Serratia marcescens, Enterococcus faecalis, and Providencia rettgeri. Sequencing of the 16S ribosmonal DNAs, selected on the basis of DGGE profiling, enabled identification of bacterial species not identified using culturable technology. This study is the first to identify biofilms in the chronic wounds of horses. In addition, this study also demonstrated the importance of combining DGGE-PCR with culture techniques to provide better microbiology analysis of chronic wounds.