The effect of whole milling and fractionation of wheat grains from the 2017 and 2018 harvests on the mycotoxin transfer into milling products and the influence of microbial fermentation of such products on mycotoxin levels were analysed. Additionally, the identification and occurrence of fungal contamination in grains with different mycotoxin contamination levels was evaluated. The mycotoxin contents were determined by chromatography (HPLC-TOF-HRMS). The distribution of mycotoxins in wheat wholemeal fractions depended on wheat kernel contamination level. The flour fractions contained zearalenone (ZEN) from 59 to 92%, and deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) at levels on average 60% of those in unprocessed wheat grains. The highest contents of enniatin B (ENNB) (84–86%) and ENNB1 (80–99%) were distributed within the flour fractions. Prolonged sourdough fermentation (48 h) allowed a reduction in DON content in the sourdough by 44–69% and a removal of 15-acetyldeoxynivalenol (15-AcDON), alternariol (AOH), deoxynivalenol-3-glucoside (D3G), toxins H-2 and HT-2, while the enniatin removal levels were in a range 5–70%, with the complete removal of only ENNB1. The study on the retention of mycotoxins during secondary grain processing (sourdough fermentation) is important for the risk assessment of wheat-based products.