In the present study, mycobiota and natural occurrence of aflatoxin B₁ (AFB₁) in pellet feed and feed ingredients used in a feed manufacturing plant for rainbow trout nutrition was investigated. The samples were cultured on the standard isolation media for 2 weeks at 28 ºC. Identification of fungal isolates was implemented based on the macro- and microscopic morphological criteria. AFB₁ was detected using high performance liquid chromatography (HPLC). Based on the results obtained, a total of 109 fungal isolates were identified of which Aspergillus was the prominent genus (57.0%), followed by Penicillium (12.84%), Absidia (11.01%) and Pseudallscheria (10.10%). The most frequent Aspergillus species was A. flavus (60.66%) isolated from all feed ingredients as well as pellet feed. Among 37 A. flavus isolates, 19 (51.35%) were able to produce AFB₁ on YES broth in the range of 10.2 to 612.8 µg/g fungal dry weight. HPLC analysis of trout feed showed that pellet feed and all feed ingredients tested except gluten were contaminated with different levels of AFB₁ in the range of 1.83 to 67.35 µg/kg. Unacceptable levels of AFB₁ were reported for feed including soybean, fish meal and wheat. These results indicate the importance of AF contamination of trout feed in amounts higher than the acceptable level as a risk factor for fish farming production.