In estuarine ecosystems, river run-off strongly influences community composition and species interactions. Although microorganisms play key roles in the functioning of these ecosystems, microbial planktonic community structure and its interaction patterns under river influence still remain poorly understood and their multisite and temporal variabilities are often overlooked. The microorganisms' small size and the difficulty to culture them under controlled conditions make the direct observation of interactions challenging. Co-occurrence networks, inferred from eDNA data and amplicon sequencing, have been commonly used to uncover potential interactions. To gain further insights into the spatiotemporal variability of microbial associations (bacteria and protists) across estuarine gradient influenced by run-off, impacted and non-impacted waters were sampled fortnightly over a 3 years period (2020-2023) across four French ecosystems. Sample-specific networks were built and their topological properties were analyzed with eight metrics. Network's structure varied among ecosystems and along the in-off shore gradients of each site. Despite pronounced spatial (between sites) and seasonal variability, a consistent pattern emerged, notably revealing a decrease in microbial networks complexity along the land-sea gradient. This greater network complexity in freshwater influenced waters might imply a higher potential for cooperation and/or competition for resources among microorganisms in highly disturbed ecosystems. Distinct microbial taxa were found to be linked either to impacted or non-impacted networks, underlying their major role in ecological interactions in estuaries.