Recent literature indicates that the elemental sulfur occurring in organic extracts of sediment samples can be toxic to the bacterium Vibrio fischeri, used in standard Microtox® bioassays. This observation was tested by means of the solvent extraction of 14 freshwater sediment samples from rivers tributary to Lake Geneva (Switzerland–France), measuring both Microtox toxicity and the elemental sulfur concentration of the extracts. Aliquots of these sediment extracts were further treated to remove the sulfur by adding acid-activated copper to the crude extracts; for 18 h in one case, and for 116 h in an other. The results were a significant amount of the observed acute toxicity in the Microtox assay of 81% of sample extracts (n = 42, crude and after cleanup) was due to elemental sulfur, and despite a median decrease of 99.1% of elemental sulfur in the extracts subject to a 116-h cleanup, sulfur toxicity was not completely excluded for 57% (8/14) of the samples. Clearly, the Microtox methodology needs to be amended to more accurately assess the potential impact of organic pollutants in sediments when solvent extracts are used. This will help to cut down on costly and unnecessary remedial actions.