ABSTRACTIn the past couple of decades, applications of environmental nucleic acids (eDNA and eRNA) analysis methods have expanded rapidly into various research fields. Recently, the World Organisation for Animal Health presented guidelines for the use of eDNA as a biomonitoring tool for disease surveillance in aquaculture. In this paper, the eDNA profile of Yersinia ruckeri was monitored over the course of experimentally induced red mouth disease in rainbow trout. Before and after mortality started, the fish pathogen burden, clinical signs, and immune response were compared to the pathogen burden in the water. Two different concentrations of bacteria were used for the infection, and the effects of handling stress on the fish's immune response were evaluated. In the higher concentration of bacteria, the eDNA profiles from Y. ruckeri were significantly higher compared to control at 3 days post‐infection (DPI) whereas the first clinical signs of the disease were reported at the first sampling point (1 DPI), outperforming the eDNA profiles. The first mortalities were recorded at 5 DPI. The handling protocol has increased mortality in fish infected with the lower concentration of bacteria without significant changes in the eDNA profile of Y. ruckeri. The eDNA profiles were significantly correlated with most of the immune genes investigated in this study in the higher concentration of bacteria, and they were primarily involved in the inflammation process (il‐6, il‐8, il‐10) and acute response (saa, hep). The use of environmental DNA is a cost‐effective and noninvasive approach for the biomonitoring of disease risks in aquaculture. Nevertheless, this study highlighted eDNA application limitations when the immune status of the fish is altered. A combined approach with eDNA and eRNA should be investigated in future infection experiments to evaluate if eRNA from the host could provide information about the immune status and the risk of increased mortality.