The assessment of freshwater ecosystem health is essential for sustainable water management amid increasing environmental stress. Aquatic macroinvertebrates and fishes serve as key bioindicators, providing integrated insights into the impacts of water quality and habitat degradation. This study investigated the spatial and seasonal patterns of aquatic invertebrate and fish communities in relation to water quality variation in two contrasting sites of the Nile Valley, Egypt (Kafr Saad and Abu Rawash areas). Sampling was conducted across different freshwater habitats, including canals, irrigation channels, ditches, and pools. A total of 73 aquatic taxa belonging to 30 families and 11 orders were recorded. Community composition and total abundance differed markedly between sites, with the Kafr Saad area exhibiting higher diversity and a greater representation of pollution-intolerant taxa, whereas pollution-tolerant groups dominated Abu Rawash. Seasonal variation significantly influenced aquatic community structure, with higher abundance and diversity observed during the warmer season. Fish taxa, particularly Poeciliidae, were more prevalent in habitats characterized by normal and good water quality. Canonical correspondence analysis (CCA) revealed that plant communities, temperature, nitrate concentration, and electrical conductivity were the main environmental drivers structuring aquatic communities. Based on integrated physicochemical and biological indicators, water quality was classified as good in the Kafr Saad area and moderately good in the Abu Rawash area. These findings highlight the value of combining aquatic macroinvertebrates and fishes in spatiotemporal biomonitoring frameworks for freshwater quality assessment in the Nile Valley.