Mycotoxins perturb the gut microbiota performance. Bioactive compounds have been recently used as a new food strategy to diminish mycotoxins bioaccessibility and prevent their toxic effects on human and animal health. Male and female Wistar rats were exposed orally to twelve different diets containing aflatoxin B1 (AFB1) and/or ochratoxin A (OTA) with or without fermented whey (FW) and pumpkin (P) for 28 days. Fecal microbiota using 16S rRNA gene sequencing and subsequent metagenomics analysis were analyzed to study the effect of 28-day exposure through diet of contaminated and enriched feed. QIIME 2 microbiome analysis package (version 2024.5) was used to analyze the demultiplexed data. Mycotoxins-functional ingredients combination contributed more to microbial phylogenetic faith alpha-diversity rather than the functional ingredients alone, while the same combination reported a microbial alpha-diversity enhancement in comparison to the mycotoxins alone. Proteobacteria phylum was reduced in rat samples fed with contaminated diets (AFB1, OTA, and AFB1+OTA), while there was an increase-although not in all groups-when adding the functional ingredients. The main difference between the sexes was found in FW+AFB1+OTA group, with males (25%) showing higher % of Proteobacteria than females (1.86%). Phylogenetic diversity faith only focuses on microbial genetic (dis)similarity, not considering the biological function. Morganella morganii, a Proteobacteria found in some groups presents anticancer activity, but it is also related to inflammatory bowel disease and colorectal cancer. To sum up, both mycotoxins and functional ingredients trigger changes in the microbiota profile of Wistar rats in a sex-specific manner.