Fjords are sensitive areas affected by climate change and can act as a natural laboratory to study microbial ecological processes. The Chilean Patagonian fjords (41–56°S), belonging to the Subantarctic ecosystem (46–60°S), make up one of the world’s largest fjord systems. In this region, Estuarine Water (EW) strongly influences oceanographic conditions, generating sharp gradients of oxygen, salinity and nutrients, the effects of which on the microbial community structure are poorly understood. During the spring of 2017 we studied the ecological patterns (dispersal and oceanographic factors) underlying the microbial community distribution in a linear span of 450 km along the estuarine-influenced Chilean Patagonian fjords. Our results show that widespread microbial dispersion existed along the fjords where bacterioplankton exhibited dependence on the eukaryotic phytoplankton community composition. This dependence was particularly observed under the low chlorophyll- a conditions of the Baker Channel area, in which a significant relationship was revealed between SAR11 Clade III and the eukaryotic families Pyrenomonadaceae (Cryptophyte) and Coccomyxaceae (Chlorophyta). Furthermore, dissolved oxygen and salinity were revealed as the main drivers influencing the surface marine microbial communities in these fjords. A strong salinity gradient resulted in the segregation of the Baker Channel prokaryotic communities from the rest of the Patagonian fjords. Likewise, Microbacteriaceae, Burkholderiaceae and SAR11 Clade III, commonly found in freshwater, were strongly associated with EW conditions in these fjords. The direct effect of EW on the microbial community structure and diversity of the fjords exemplifies the significance that climate change and, in particular, deglaciation have on this marine region and its productivity.