Macroinvertebrate communities were investigated along a gradient of heavy industrial and municipal pollution in the highland Bílina River (Czech Republic). Physico-chemical determinants and ions were monitored and community analysis performed focusing on taxonomic composition, ecological functioning (feeder and dweller guilds) and water quality metrics, including saprobity index, BMWP and diversity. Impacted sites differed significantly from reference and from recovered stretches. Chemical data revealed two main pollution factors, (1) a “salinity determinant”, described best by conductivity and SO₄²⁻, and (2) an “organic pollution determinant”, represented best by O₂ concentrations and NO₂⁻, all varying locally and temporally. Some metrics and taxa showed significant correlations to abiotic parameters. Functional communities showed a stronger relationship to the “organic pollution determinant”, suggesting that elevated organic pollution had a dominating influence on functional community metrics; though other variables may also have an influence in this multistress environment. On the other hand, there were indications that the taxonomic community was more influenced by ion concentrations (“salinity determinant”). The gradient from reference sites to polluted sites was weaker in the final sampling campaign. The results presented here can be used as a reference for assessing future changes in environmental impact from pollution, being finer and more detailed than assessment according to the EU’s WFD.