Background: Renibacterium salmoninarum and Mycobacterium sp. are important bacterial pathogens of fish. R. salmoninarum is the causative agent of bacterial kidney disease, a Gram-positive bacterium mostly known for causing chronic infections in salmonid fish, while multiple species belonging to the Mycobacterium genus have been associated with mycobacteriosis in fish as well as in human. The objective of this study was to determine the prevalence of these two bacterial pathogens in populations of wild brown trout (Salmo trutta fario) in four rivers (Kamp, Wulka, Traun and Ybbs) in Austria. Results: A total of 457 kidney samples were examined for both bacterial agents using nested and conventional PCR as well as bacterial cultivation on KDM-2, histological examination and immunohistochemistry. Molecular evidence showed an estimated prevalence level of 0.94% for R. salmoninarum in 2017 while the bacterium could not be detected in 2018 and histology showed signs consistent with a low-level chronic inflammation in the kidney of infected fish. Similarly, no fish were found positive for Mycobacterium in 2017 but in 2018, the prevalence was found to be 37.03% in the Kamp river (4.08% across all rivers). The sequencing data confirmed that these fish carried Mycobacterium sp. although the precise species of Mycobacterium could not be ascertained. Conclusions: This survey constitutes the first insight into the prevalence rate of R. salmoninarum and Mycobacterium sp. in wild brown trout (Salmo trutta fario) populations in Austria. Both of these pathogens were only detected in the summer months (June and July), which might suggest that the stress linked to increased water temperature could act as stressor factor and contribute to the outbreak of these diseases. The age of the fish might also play a role, especially in the case of Mycobacterium sp. as all the infected fish were in their first summer (June).