Microcystins are toxins produced by cyanobacteria. They occur in aquatic systems across the world and their occurrence is expected to increase in frequency and magnitude. As microcystins are hazardous to humans and animals, it is essential to understand their fate in aquatic systems in order to control health risks. While the occurrence of microcystins in sediments has been widely reported, the factors influencing their occurrence, variability, and spatial distribution are not yet well understood. Especially in shallow lakes, which often develop large cyanobacterial blooms, the spatial variability of toxins in the sediments is a complex interplay between the spatial distribution of toxin producing cyanobacteria, local biological, physical and chemical processes, and the re-distribution of toxins in sediments through wind mixing. In this study, microcystin occurrence in lake sediment, and their relationship with biological and physicochemical variables were investigated in a shallow, eutrophic lake over five months. We found no significant difference in cyanobacterial biomass, temperature, pH, and salinity between the surface water and the water directly overlying the sediment (hereafter ‘overlying water’), indicating that the water column was well mixed. Microcystins were detected in all sediment samples, with concentrations ranging from 0.06 to 0.78 µg equivalent microcystin-LR/g sediments (dry mass). Microcystin concentration and cyanobacterial biomass in the sediment was different between sites in three out of five months, indicating that the spatial distribution was a complex interaction between local and mixing processes. A combination of total microcystins in the water, depth integrated cyanobacterial biomass in the water, cyanobacterial biomass in the sediment, and pH explained only 21.1% of the spatial variability of microcystins in the sediments. A more in-depth analysis that included variables representative of processes on smaller vertical or local scales, such as cyanobacterial biomass in the different layers and the two fractions of microcystins, increased the explained variability to 51.7%. This highlights that even in a well-mixed lake, local processes are important drivers of toxin variability. The present study emphasises the role of the interaction between water and sediments in the distribution of microcystins in aquatic systems as an important pathway which deserves further consideration.