The chronological framework for Neanderthal occupation and demise across Europe continues to be debated. In particular, there is still uncertainty regarding the nature, timing and regional expressions of the Middle to Upper Palaeolithic transition associated with the disappearance of Neanderthals and the broader expansion of modern human populations in Europe around 42–40 thousand years ago (ka). The geographical and chronological distribution of Neanderthal populations also remains difficult to evaluate owing to the practical challenges of directly dating human fossils at many sites, and the fact that a large proportion of Neanderthals sites lie close to, or well-beyond, the limits of radiocarbon dating. Cova del Gegant – one of the few sites in north-eastern Iberian Peninsula to yield Neanderthal fossil remains, associated Mousterian archaeological layers, and occupations related to the Middle and Upper Palaeolithic transition – is a key locality for informing these ongoing debates. Here we provide a comprehensive chronological framework for the Cova del Gegant site using multiple radiometric dating techniques (uranium-thorium (U–Th), radiocarbon and luminescence dating), sedimentological and micromorphological analyses, and Bayesian modelling. This integrated chronostratigraphic approach enables us to reliably reconstruct site formation processes and history, and undertake improved correlations with other sites regionally. The results allow us to sub-divide the Cova del Gegant sequence into three sections spanning ∼94 ka to ∼32 ka, namely: a Middle Palaeolithic sequence covering ∼94–59 ka, a Châtelperronian/Aurignacian section spanning ∼43–39 ka, and a Late Aurignacian/Gravettian section spanning ∼34–32 ka. The Neanderthal fossil remains accumulated in the cave between the end of Marine Isotope Stage (MIS) 5/MIS 4 and the beginning of MIS 3, during two different events dated to ∼72–67 ka and ∼60–52 ka. The chronological framework for Cova del Gegant is in accordance with that reported for other Middle and Upper Palaeolithic sites in north-eastern Iberian Peninsula, and reveals a record of successive human occupation coinciding with a period of progressive global cooling and lowering sea levels (end of MIS 5 through to MIS 2). Sedimentological evidence points to the emergence of a coastal platform in front of the cave and indicates that local palaeoenvironmental conditions likely benefited human displacements along the littoral margin, and favoured repeated occupation of the cave during the Late Pleistocene.