Wound healing and tissue regeneration are essential mechanisms to ensure the survival and health of any organism. Despite this, only a few studies have been devoted to study tissue regeneration during wound healing in fish. Reactive oxygen species (ROS), in particular hydrogen peroxide, play an important dual role both for promoting tissue repair, but also for eradication of pathogens. This study aims at dissecting the contribution of PAMPs (using β-glucan) and DAMPs in the respiratory burst response of carp head kidney-derived leukocytes, and address their contribution to wound healing processes. Consistent with a pathogen eradication strategy, ROS responses to PAMP stimulation (β-glucan) was fast, vigorous and highly dominated by production of superoxide anion. In contrast, stimulation with DAMPs led to a slow, subtle but long-lasting production of oxygen radicals dominated by hydrogen peroxide. Using an in vitro model of scratch-wounded CCB fibroblast cell cultures and a novel PhotoID proliferation assay, stimulation with low and continuous levels of hydrogen peroxide (5μM) led to a slight increase in the percentage of wound recovery and thus promoted wound closure. In contrast, high doses of hydrogen peroxide (300μM) impaired fibroblast scratch-wound recovery and caused cell death. These results elucidate the capacity of hydrogen peroxide to influence the fate of tissue regeneration through the establishment of environments suitable for promoting either tissue regeneration or oxidative stress and thereby potential tissue damage. Direct in vitro stimulation with β-glucans did not impact fibroblast scratch-wound recovery, which further suggests that interaction with tissue-resident leukocytes or other components of the fish immune system are required to induce fibroblast proliferation and thus for the accelerated wound healing promoted by β-glucan stimulation.