Mycotoxins are toxic secondary metabolites produced by various fungi that can contaminate food crops, which, in turn, may lead to human exposure. Chronic exposure to mycotoxins can cause adverse health effects including reproductive and developmental toxicity. Pregnant women and their foetuses present a vulnerable group for exposure to mycotoxins that can cross the placenta. Human biomonitoring of mycotoxins provides a real-life approach to estimate internal exposure. In this pilot study, 24-h urine samples from 36 pregnant Dutch women were analysed for aflatoxin M1 (AFM1), total deoxynivalenol (DON), de-epoxy-deoxynivalenol (DOM-1), total zearalenone (ZEN), total α-zearalenol (α-ZEL), total β-zearalenol (β-ZEL) and total zearalanone (ZAN), where ‘total’ refers to mycotoxins and their conjugated forms. Serum samples from these women were analysed for fumonisin B1 (FB1) and ochratoxin A (OTA). All samples were measured using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The most prevalent mycotoxins were total DON, total ZEN and OTA, with a detection frequency of 100%. DOM-1, total α-ZEL and total β-ZEL were detected but to a lesser extent, while AFM1, total ZAN and FB1 were undetected. Median concentrations were 4.75 μg total DON/L, 0.0350 μg DOM-1/L, 0.0413 μg total ZEN/L, 0.0379 μg total α-ZEL/L, 0.0189 μg total β-ZEL/L, and 0.121 μg OTA/L. The calculated median concentration for total ZEN and its metabolites was 0.105 μg/L. Based on two separate risk assessment approaches, total DON exposure in this group was considered to be of low concern. Similarly, exposure to total ZEN and its metabolites in this group was of low concern. For OTA, the risk of non-neoplastic effects was of low concern based on exposure in this group, and the risk of neoplastic effects was of low concern in the majority of participants in this group. The findings of this pilot study confirm the presence of mycotoxins in the urine and serum of pregnant Dutch women, with total DON, total ZEN, and OTA most frequently detected. ...