Aims: Rhizogenic Agrobacterium strains are the causative agent of hairy root disease (HRD), an increasing problem in the hydroponic cultivation of tomato and cucumber in Europe. A previous study has demonstrated that different lineages of rhizogenic agrobacteria are able to form biofilms. Although hydrogen peroxide (H2O2) is a frequently used biocide in hydroponic systems, until now its effectiveness to remove rhizogenic agrobacteria has not been unequivocally demonstrated. Therefore, the main objective of this study was to assess the efficacy of H2O2 in controlling Agrobacterium-containing biofilms. Methods and Results: Using lab-scale experiments, we found a huge variation between different rhizogenic Agrobacterium strains in EC50 values, ranging from 18.8 to 600 ppm H2O2, representing the lowest and highest concentration tested respectively. Using pilot-scale experiments in which different H2O2 concentrations were tested, treatment with 25 ppm H2O2 was found to be ineffective. In contrast, treatment with 50 ppm significantly affected a catalase-negative Agrobacterium population, while a catalase-positive population was only marginally affected. For the catalase-positive Agrobacterium population, a treatment of 100 ppm H2O2 was required to be effective. Finally, H2O2 treatment of HRD in two commercial greenhouses was monitored, and showed that the H2O2 concentration decreased considerably towards the end of the irrigation circuits. Further, a clear correlation was found between the actual concentration of H2O2 and the incidence of HRD. Conclusion: We showed that H2O2 may be effective to reduce biofilm formation by rhizogenic bacteria. Nevertheless, it was clear from our results that the required H2O2 concentration depends on the particular Agrobacterium strain(s) present in the greenhouse. Significance and Impact of the Study: This is the first study that examined the effectiveness of H2O2 to control HRD in hydroponic systems, and the effect of catalase activity on H2O2 effectiveness. Our study has direct relevance for the highly intensive horticultural sector.