The open-air site of Nahal Hesi, located on northwestern Negev desert fringe of Israel, is one of the few primary-context late Acheulian localities in the Levant. Late Acheulian sites in the Levant rarely provide faunal assemblages and radiometric ages, leaving large gaps in our knowledge of the Lower Paleolithic palaeoenvironment, ecology, subsistence and chronology. Here we present a new geo-chronological study conducted at the Nahal Hesi site. The major objectives were to describe the geological and archaeological stratigraphy, assign a numerical age to the Acheulian artifact-bearing deposit by TT-OSL and pIR-IR290 dating, and reconstruct the local palaeoenvironment during the human occupation at the site. Geological and microfaunal observations suggest that late Acheulian hominins at Nahal Hesi exploited marshy environments. The limited spatial and vertical extent of the artifact-bearing deposit and the presence of artifacts only in context of marsh-like sediments, highlight the utilization of short-lived water bodies by the hominins. The combined TT-OSL and pIR-IR290 methods provide an average age of 430 ± 35 ka for the studied marsh-like deposits. The study indicates that the Middle Pleistocene in the area of the Nahal Hesi site is characterized by lengthy cycles of deposition and erosion of fine-grained sediments associated with the semi-arid Nahal Shiqma drainage system. Marshy sediments could be the result of full or partial blockage of the Shiqma system, probably by coastal dune ridges, that led to formation of standing water bodies during the Middle Pleistocene.