Abstract Hydrogen peroxide (H2O2) treatment is an alternative for disinfection in aquaculture, which may be advantageous as it dissociates and disinfects while increasing water oxygen concentration. Yet, accurate dosing remains undeveloped in Recirculating Aquaculture Systems (RAS). Dosage requirements can depend on organic burden, stocking density, feeding frequency, salinity, temperature and biofilter performance. The present case study investigated the dual effect of H2O2 application for oxygen enrichment and disinfection when continuously applied to a RAS rearing European seabass. H2O2 addition equivalent to 2.4 and 15.8 H2O2 mg L−1 were applied for 4 h per day in three 5-days experiments. H2O2 was injected at the inlet of protein skimmer and/or the rearing tanks in or without combination with traditional disinfection methods. Water microbial load and oxygen saturation were determined, along with stress markers glucose and cortisol in blood plasma of fish. Doses of 15.8 mg L−1 H2O2 steadily increased oxygen levels in holding tank water from ∼50 % to over 100 % saturation while reducing microbial load (from 604.4 CFU ml−1 in the rearing tanks before dosing to 159.8 CFU ml−1 after application), achieving suitable conditions for commercial fish densities in RAS. The doses used had negligible impact on biofilter performance and did not affect the fish in terms of stress. Overall results indicate H2O2 is effective for disinfection and oxygenation of RAS systems when applied at appropriate dosage and we recommend the protein skimmer as the safest position in order to protect the bacterial community of the biofilters and the reared fish.