Banwari Trace, a well-stratified shell midden located in southeastern Trinidad, provides the oldest known archaeological evidence of human settlement in the West Indies and has been crucial to our understanding of the initial peopling of the greater Caribbean region. Detailed excavation profile descriptions, soil and faunal analyses, accelerator mass spectrometry radiocarbon and optically stimulated luminescence dating, and stable carbon isotope analyses provide an accurate chronology and paleoenvironmental framework for the natural and anthropogenic depositional history of this significant archaeological site. Our findings support the recognition of three Middle Holocene strata at Banwari Trace, which represent significant periods of midden deposition and environmental change at: ~7800–7900 cal BP (Level 3); ~6900–7400 cal BP (Level 2); and ~5500–6200 cal BP (Level 1). Stable carbon isotope analyses show the landscape was dominated by C3 vegetation throughout the Middle Holocene with a possible drying episode near the end of the Middle Holocene climatic optimum. Cedrosan potsherds discovered in the uppermost 25 cm (Level 0) suggest that a Late Holocene radiocarbon age of ~2770–2200 cal BP for charcoal from this stratum is valid and possibly contemporary with an apparently intrusive human burial recovered in 1971 at a depth of ~20 cm.