In this article, using the Hawkesbury-Nepean River as a case study, the spatial and temporal trends of water quality variables over three sampling surveys in a peri-urban situation are examined for their effect on benthic macroinvertebrate communities and phytoplankton communities and whether phytoplankton and benthic macroinvertebrate species can be used as indicators for river health assessment. For this, the authors monitored the spatial and temporal difference of 10 water quality parameters: temperature, turbidity, pH, dissolved oxygen, electrical conductivity, oxidation reduction potential, total nitrogen, total phosphorus, manganese, and suspended solids. The variability in water quality parameters clearly indicated a complex pattern, depending on the season (interaction p = 0.001), which highlighted how the river condition is stressed at multiple points as a result of anthropogenic effects. In particular, the downstream locations indicated an accumulation of nutrients, the presence of increased sediments, and phytoplankton related variables such as total counts, bio-volumes, chlorophyll-a, and total phosphorus. The patterns of phytoplankton communities varied in a complex way depending on the season (interaction p = 0.001). Abundances of phytoplankton were also found in low concentrations where the water column is not severely disturbed by flow and tide. However, when the water clarity drops resulting from tidal cycles, inflows from tributaries, and intense boating activities, the phytoplankton abundances also increased considerably. On the other hand, benthic macroinvertebrates compositions were significantly different between locations (p = 0.001) with increased abundances associated with upstream sites. Aphanocapsa holsatica and chironomid larvae appeared as the important indicators for upstream and downstream site differences in water quality. Water temperature influenced the phytoplankton community pattern (ρ(w) = 0.408), whereas pH influenced the benthic macroinvertebrate community pattern (ρ(w) = 0.437). The findings of this study provide valuable insights into the interactions of water quality parameters on biotic assemblages and to the extent that benthic macroinvertebrates and phytoplankton assemblages are suitable as indicators for monitoring and assessing peri-urban river health.