Histophilus somni is an opportunistic pathogen causing respiratory, genitourinary and generalized infections in cattle. An important virulence factor is its ability to produce a biofilm. The aim of this work was to confirm that H. somni Hsp60 (Gro-EL) is a constituent of the biofilm produced by this bacterium in vitro and to check whether or not the presence of a specific antibody within the culture medium can inhibit biofilm production. Biofilm production by H. somni cultured in vitro was confirmed by crystalline violet staining. The presence of Hsp60 in the biofilm was confirmed by using specific antibodies produced in a mouse and goat hyperimmunized with H. somni recombinant Hsp60 (rHsp60). Large complexes of biofilm stained with Hsp60 antibodies were microscopically detected. This indicates that the Hsp60 protein is a common constituent of the biofilm produced by H. somni in vitro. In a second experiment, mouse serum containing anti-H. somni rHsp60 antibodies was added to an H. somni culture. It was found that the presence of anti-rHsp60 antibodies in the culture medium inhibited biofilm production in vitro. Only small biofilm particles were seen in the presence of the specific antibody, whereas in control cultures (without specific antiserum) large biofilm complexes were produced. The results indicate that antibodies specific to Hsp60 may be useful for preventing H. somni biofilm formation in vitro. If this also occurs in vivo, it may be helpful for eradicating H. somni infection in cattle through the elimination of carriers. Further in vivo studies are needed to confirm this idea.