The effective conditions of glutaraldehyde, chloramine-T, bronopol, Incimaxx Aquatic® and hydrogen peroxide as some biocides commonly used by the aquaculture industry were investigated against F. psychrophilum in sanitization of rainbow trout eyed eggs. Bacteriostatic tests as well as bactericidal tests using ethidium monoazide bromide PCR assays were conducted in vitro on Flavobacterium psychrophilum while impacts of chemical treatments were studied in vivo on 240 [°C × days] rainbow trout eyed eggs. A 20-min contact time with bronopol (up to 2,000 ppm), chloramine-T (up to 1,200 ppm), glutaraldehyde (up to 1,500 ppm), hydrogen peroxide (up to 1,500 ppm) or with Incimaxx Aquatic® (up to 185 ppm, eq. peracetic acid) was effective against F. psychrophilum and did not affect the eyed eggs/fry viability. Collectively, the data obtained here clearly demonstrate that concentrations and duration of treatments commonly used to sanitize eyed eggs are widely overestimated in their effectiveness against F. psychrophilum. The new treatment conditions with the five studied biocides are bactericidal for F. psychrophilum and safe for rainbow trout eyed eggs. In this work, we developed an experimental approach to test some chemicals against fish pathogens to assist fish farmers in the effective and safe disinfection of eyed eggs.