Groundwater microbial communities exhibit pronounced vertical and spatial structuring driven by physicochemical gradients. Here, we investigated microbial assemblages across surface and subsurface layers of three groundwater wells distributed along a 1.26 km transect in the Wadi Awja aquifer system (Jeddah, Saudi Arabia) using high-throughput 16S rRNA gene amplicon sequencing. Across all samples, Pseudomonadota (Proteobacteria) dominated community composition, accounting for ~50–65% of surface assemblages and increasing to ~90% in deeper strata, indicating strong vertical selection. This depth-associated enrichment coincided with reduced community evenness and the prevalence of metabolically versatile, facultatively anaerobic taxa. Although Actinomycetota, Bacteroidota, and Planctomycetota contributed substantially to overall diversity, their relative abundances declined with depth, reinforcing the dominance of Proteobacteria under suboxic conditions. Notably, members of Enterobacteriaceae, particularly Escherichia spp., were consistently enriched in deeper layers, coinciding with simplified community structures. Collectively, these results demonstrate that groundwater microbial communities undergo sharp redox-associated ecological transitions over short spatial scales, emphasizing the role of localized hydrogeochemical heterogeneity in shaping subsurface microbial assemblages.