Trace metal analysis and speciation were performed at the sediment–water interface of Vidy Bay (Lake Geneva, Switzerland). This bay is impacted by hazardous compounds released via the sewage effluent of a major wastewater treatment plant (WWTP). Sediment cores and overlying water were sampled simultaneously at 12 sites characterized by contrasting sediment surface characteristics (color, methanogenic activity, bacterial mat) using corers deployed from a MIR submarine or research boat. The concentrations of trace metals in particulate form in the sediment and dissolved in the interstitial water, as well as the particulate, colloidal and dynamic fractions of trace metals in the overlying water were determined by combining an in situ and laboratory multi-method analytical approach. The results indicate differences in trace metal speciation in the sediment and overlying water at the 12 investigated sites. The observed differences were found to be more correlated to bacterial community, abundance, type and activity than to distance from the WWTP sewage outlet.