Microorganisms drive essential biogeochemical processes in aquatic ecosystems and are sensitive to both salinity and hydrological changes. As climate change and anthropogenic activities alter hydrology and salinity worldwide, understanding microbial ecology and metabolism becomes increasingly important for managing aquatic ecosystems. Biogeochemical processes were investigated on sediment microbial communities during a significant flood event in the hypersaline Coorong lagoon, South Australia (the largest in the Murray-Darling Basin since 1956). Samples from six sites across a salinity gradient were collected before and during flooding in 2022. To assess changes in microbial taxonomy and metabolic function, 16S rRNA amplicon sequencing was employed alongside untargeted liquid chromatography–mass spectrometry (LC-MS) to assess changes in microbial taxonomy and metabolic function. Results showed a decrease in microbial richness and diversity during flooding, especially in hypersaline conditions. Pre-flood communities were enriched with osmolyte-degrading and methanogenic taxa, alongside osmoprotectant metabolites, such as glycine betaine and choline. Flood conditions favored taxa such as Halanaerobiaceae and Beggiatoaceae, inducing inferred metagenomic shifts indicative of sulfur cycling and nitrogen reduction pathways, while also enriching a greater diversity of metabolites including Gly-Phe dipeptides and guanine. This study demonstrates that integrating metabolomics with microbial community analysis enhances understanding of ecosystem responses to disturbance. These findings suggest microbial communities rapidly change in response to salinity reductions while maintaining key biogeochemical functions. Such insights are valuable for ecosystem management and predictive modelling under environmental stressors such as flooding.