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Aquaculture is a growing industry where facilities require robust strategies to help prevent the spread of infectious diseases. Using effective biocides as disinfectants in biosecurity plans is essential to implement a comprehensive safety program. Although the majority of high-level commercial biocides acting effectively on the free-living planktonic pathogens seldom are also helpful on the more complex biofilm structures. Here, we evaluated the antimicrobial time-efficacy of three disinfectant biocides, Virkon™Aquatic (VirA), peracetic acid (PerA), and hydrogen peroxide (HydP) upon Vibrio anguillarum, Vibrio harveyi, Vibrio alginolyticus, and Photobacterium damselae subspecies piscicida against their both living strategies. By using the minimum inhibitory and bactericidal concentrations sufficient on the free-living planktonic state (MIC; MBC) and biofilms (MBIC; MBEC), we determined in vitro the susceptibility of each bacterial strain against three different individual concentrations of VirA, PerA, and HydP were added at 1, 5, and 10 minutes intervals. We found that PerA and VirA had the highest bactericidal efficacies against all bacteria' free-living planktonic state and biofilm. Kinetically, PerA achieved a significantly quicker positive result in both cases and did not vary among strains, while the weakest HydP required longer than 10 min to act effectively. Besides, we conducted a short in vivo safety trial using juvenile Gilthead seabream (Sparus aurata). Disinfectants were poured each at the suggested MIC in the water of triplicate tanks per group holding fish. Mortality started as early as 12 h post-addition in the VirA group, while PerA and HydP did it so much later. Whatever the case, all groups achieved no more than 50% mortality after 24 h, showing the affordable risk these disinfectants possess for fish. Together, our results strongly support the use of biocides as disinfectants in aquaculture facilities and highlight PerA as the most effective fighting against biofilms produced by V. anguillarum, V. harveyi, V. alginolyticus, and P. damselae subsp. piscicida., {"references":["doi.org/10.1016/j.aquaculture.2020.736004"]}