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This study aimed to obtain and characterize an oil-in-water nanoemulsion (NE) loaded with an in vitro optimized bactericidal essential oil blend of 50% oregano, 40% thyme, and 10% lemongrass and to evaluate its potential at three different concentrations (0.5%, 1%, and 2%) in the inactivation of <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, and <i>Salmonella enterica</i> serotype Enteritidis inoculated in rainbow trout fillets stored at 4 °C for 9 days. Regarding the NE, the nanometric size (<100 nm) with low polydispersion (0.17 ± 0.02) was successfully obtained through ultrasound at 2.09 W/cm². Considering the three concentrations used, <i>S</i>. Enteritidis was the most susceptible. On the other hand, comparing the concentrations used, the NE at 2% showed better activity, reducing <i>S.</i> Enteritidis, <i>E. coli</i>, and <i>S. aureus</i> by 0.33, 0.20, and 0.73 log CFU/g, respectively, in the trout fillets. Thus, this data indicates that this is a promising eco-friendly alternative to produce safe fish for consumption and reduce public health risks.