The Prespa Lakes area in Greece—comprised partly of lake Great and lake Lesser Prespa and the Vromolimni pond—has a global importance for biodiversity. Although the waters show regular cyanobacteria blooms, assessments of water quality threats are limited. Samples collected in 2012 revealed scattered and low microcystin (MC) concentrations in Great Prespa (<0.2 μg MC L−1) whereas considerable spatial heterogeneity in both total chlorophyll (2.4-93 µg L−1) and MC concentrations (0.04-52.4 µg MC L−1) was detected in Lesser Prespa. In 2013, there was far less spatial variability of MC concentrations in Lesser Prespa (0.4-1.53 µg L−1), however in 2014, increased concentrations were detected near the lakeshore (25-861 µg MC L−1). In Vromolimni pond the MC concentrations were on average 26.6 (±6.4) µg MC L−1 in 2012, 2.1 (±0.3) µg MC L−1 in 2013 and 12.7 (±12.5) µg MC L−1 in 2014. In 2013, no anatoxins, saxitoxins, nor cylindrospermopsins were detected in Lesser Prespa and Vromolimni waters. Tissue samples from carps, an otter and Dalmatian Pelicans contained 0.4-1.9 µg MC g−1 dry weight. These results indicate that cyanotoxins could be a threat to the ecosystem functions of particularly Lesser Prespa and Vromolimni.