The Dender basin in Flanders (Belgium) was used as a case study to implement the European Union (EU) Water Framework Directive. During the last 5 years, ample research on pollution loads and ecological water quality has been done on the Dender River. In addition to biological sampling of macroinvertebrates and fish, automated measurement stations were also used to investigate the spatial-temporal variability of the physical-chemical water quality. This research revealed that the pollution of the Dender River is highly variable. The high nutrient loads result in severe algae blooms during summer, leading to very complex diurnal processes. In this paper, the monitoring strategy for the assessment of the biological water quality in the Dender basin has been reviewed in relation to the EU Water Framework Directive. For this, seasonal macroinvertebrate data were collected and assessed. General trends and hidden structures in these data were analysed by means of classification trees, using different inputs (seasons, river types, and subbasins). Validation of the results was obtained by applying statistical methods. Analysis about the presence and abundance of the macroinvertebrates revealed that there is a distinct difference between the biological water quality in the Dender stem river and its tributaries. There are also seasonal differences between the macroinvertebrate communities when the Dender and its tributaries are examined separately. An optimised monitoring strategy is proposed based on these results and the EU Water Framework Directive. This includes two monitoring campaigns in summer and winter every 3 years. Furthermore, a cyclic monitoring scheme was developed to minimise sampling efforts.