Benthic organisms are crucial in the regulation of the ecosystem functions. They are primary mediators of the energy transfer in marine benthic food webs. Trophic interactions between benthic nematodes and sediment bacteria across divergent environmental conditions are poorly understood. The main goal of this study was to comprehend the relation between the spatial distribution patterns and diversity of benthic microbial communities and nematode assemblages of the intertidal sediments in Sado estuary (SW, Portugal). Samples were taken from three sites with different biogeochemical sediment conditions. Microbial communities were described using metagenomic approach, while nematode assemblages were characterized using morphological identification. Bacterial communities and nematodes assemblages presented significant heterogeneity between sites (p<0.05), which were primary related with the environmental variables (e.g., organic matter deposits and gravel percentage). However, while microbiome distributional patterns were clearly in accordance with ecological conditions of three selected sites (adjusted R2Adj = 0.53), nematode assemblages were more responsive to specific sampling locations within each site, suggesting that their response is rather driven by the within site specific factors, acting at the smaller spatial scale. Our study also demonstrated that the presence of Cyanobacteria (higher relative abundance of Pleurocapsa PCC-7319) seemed to be correlated with high abundance of nematode deposit feeders Terschellingia and Sabatieria indicating possible interactions between these taxa. Our study represents a first insight into bacteria–nematodes associations underlying ecological conditions thereby providing an important baseline for the future understanding of the role that these two groups play in the benthic estuarine ecosystem.