Modern discoveries of two naturally occurring toxins that thrive in limestone-rich environments offer clues to understanding how the Styx (Mavroneri) River received its nefarious reputation in antiquity. In Greek myth, the Styx was the entrance to the Underworld. The Styx was believed to be poisonous, dangerous even to the gods who swore oaths by its waters. After the death of Alexander the Great in 323 BCE, rumors arose that he had been deliberately poisoned by the water of the Styx. This paper analyzes descriptions of the geomyth about Styx’s deadly nature in ancient Greek and Latin literature in light of modern geology and toxicology. Recent identifications of two types of lethal soil-based organisms raise the possibility that the Styx might have harbored a naturally occurring toxic fungus or bacterium in antiquity. I propose three hypotheses, which, if they could be proven, might identify the mysterious poison associated with the river and help explain why the Styx water was believed to be fatal. Keywords: Styx · Poison · Toxicology · Mythology · Alexander the Great · Toxic lichen · Calicheamicin.