The immune response in rainbow trout fry against Flavobacterium psychrophilum was elucidated using an immersion-based challenge with or without prior exposure to hydrogen peroxide (H2O2). Samples were taken from the head kidney 4, 48, 125 and 192 h after immersion, and the regulation of several genes was examined. Bacterial load was assessed based on the presence of 16S rRNA and correlated with gene expression, and the levels of specific antibodies in the blood were measured 50 days post-infection. Separately, both H2O2 and F. psychrophilum influenced gene expression, and pre-treatment with H2O2 influenced the response to infection with F. psychrophilum. Pre-treatment with H2O2 also affected correlation between gene regulation and pathogen load for several genes. A delay in antibody production in H2O2-treated fish in the early phase of infection was indicated, but H2O2 exposure did not affect antibody levels 50 days post-infection. An increasing amount of F. psychrophilum 16S rRNA was found in the head kidneys of infected fish pre-treated with H2O2 relative to the F. psychrophilum group. The results show that a single pre-treatment with H2O2 impairs the response against F. psychrophilum and may intensify infection. © 2013 John Wiley & Sons Ltd.