Mycotoxins are known for their negative impact on human and animal health as they frequently contaminate food and feed products from crop origin that are consumed by humans and animals. Furthermore, mycotoxins can leach out of plant tissue, to be transported through runoff water into nearby ponds where they can exert negative effects on aquatic organisms, such as fish, amphibians and zooplankton. The overall goal of this study was to develop a SPE-UHPLC-MS/MS method for the detection and quantification of multiple mycotoxins in amphibian breeding ponds. The method was validated and yielded acceptable within-run and between-run apparent recoveries and precision, as well as good linearity. Matrix effects (i.e. 75.7–109.6%, ≤ 17.8% RSD) were evaluated using water from 20 different ponds in Flanders, Belgium. By incorporating internal standards, overall results improved and adequate precision values (i.e. ≤ 15%) were obtained according to the EMA guideline. Additionally, extraction recovery (n = 3) was evaluated, yielding good results for all mycotoxins (i.e. 75.3–109.1%, ≤15% RSD), except for AME (i.e. 6.7 ± 0.7%), which implied the need for a matrix-matched calibration curve. Detection sensitivity was in the low nanograms per liter range. Storage stability experiments indicated that sample storage at 4 °C in amber glass bottles and analysis performed within 96 h after sampling was sufficient to avoid loss by degradation for all compounds, excluding β-ZAL and β-ZEL, for which analysis within 24 h is more indicated. The method was successfully applied to water samples originating from 18 amphibian breeding ponds situated across Flanders. Overall, enniatins B, B1 and A1 were most commonly detected at maximum concentrations of 6.9, 3.3 and 2.6 ng L−1, respectively, followed by detection of beauvericin (1.1 ng L−1 and < 1 ng L−1), alternariol monomethyl ether (< 10 ng L−1), HT2-toxin (< 40 ng L−1), zearalenone (< 25 ng L−1) and α-zearalanol (< 10 ng L−1). We believe that this method will boost further research into the dynamics and ecotoxicological impact of mycotoxins in aquatic environments.