Abstract Streptococcus equi ssp . zooepidemicus ( S. equi spp. zooepidemicus ) is an opportunistic pathogen that causes major economic losses in the swine industry in China and is also a threat for human health. Biofilm formation by this bacterium has been previously reported. In this study, we used an immunoproteomic approach to search for immunogenic proteins expressed by biofilm-grown S. equi spp. zooepidemicus . Seventeen immunoreactive proteins were found, of which nine common immunoreactive proteins were identified in planktonic and biofilm-grown bacteria. The immunogenicity and protective efficacy of the S. equi spp. zooepidemicus immunoreactive GroEL chaperone protein was further investigated in mice. The protein was expressed in vivo and elicited high antibody titers following S. equi spp. zooepidemicus infections of mice. An animal challenge experiment with S. equi spp. zooepidemicus showed that 75% of mice immunized with the GroEL protein were protected. Using in vitro biofilm inhibition assays, evidence was obtained that the chaperonin GroEL may represent a promising target for the prevention and treatment of persistent S. equi spp. zooepidemicus biofilm infections. In summary, our results suggest that the recombinant GroEL protein, which is involved in biofilm formation, may efficiently stimulate an immune response, which protects against S. equi spp. zooepidemicus infections. It may therefore be a candidate of interest to be included in vaccines against S. equi spp. zooepidemicus infections.