In contrast to numerous documented pathogens and infectious diseases of aquaculture, there is a lack of baseline data and information regarding pathogenic agents’ prevalence in wild marine fish populations. This study focused on two common fish pathogenic microorganisms, namely Mycobacterium species and Vibrio species, both of which are known to be major causes of fish loss, occasionally to the extent of being a limiting factor in fish production. Both microorganisms are known as zoonotic agents. In total, 210 wild marine indigenous and Lessepsian fish from four different species from the eastern Mediterranean Sea were sampled and tested for Vibrio species and Mycobacterium species during a two-year period (2016–2017). Using PCR with 16S rRNA primers, we detected different strain variations of Mycobacterium species and Vibrio species and, based on the sequencing results, the overall prevalence for Vibrio species in wild fish in 2016 was significantly higher compared to 2017. No significant difference was detected for Mycobacterium species prevalence in wild fish between 2016 and 2017. In addition, 72 gilthead seabream (Sparus aurata) from an Israeli offshore marine farm were also examined during the two-year period (2017–2018). The results suggest that Mycobacterium species prevalence was significantly higher in 2018, while in 2017 there was no positive results for Mycobacterium species. In addition, there was no significant difference between both years in regard to the prevalence of Vibrio species for maricultured fish. These results highlight the necessity of continuous molecular monitoring in order to evaluate the prevalence of pathogenic microorganisms in both wild and cultured fish populations.