Marine insertion indicators in the Vitoria estuary (ES) revealed relative variations in sea level during the Holocene in three sediment cores. Sedimentological, geochemical (C/N ratio), and paleontological (shells and palynomorphs) analysis and 14C dating associated five sedimentary facies to different estuarine deposits. A C/N ratio <10 at the core base indicated organic matter of marine origin. Moving up the cores to 110–150 cm, an abrupt increase in C/N to 26–63 in every core suggests the sudden entry of higher plants into the estuary, potentially the moment sea level retreated. High continental (10,743 palynomorphs/g) and low marine palynomorph concentrations (323 palynomorphs/g) suggest a primarily continental source even during transgression and at high sea level. Around 8973 cal BP, an open bay already existed in the region of Vitoria. Sea level potentially exceeded the current level around 7110 cal BP. The transgressive maximum was at 5567 cal BP. Marine insertion indicators, such as marine shells, low C/N ratios and foraminiferal linings, did not always respond directly to sea level oscillations. These discrepancies probably result from lateral variations in sedimentary deposits from transport patterns and from variations in organic matter and palynomorph preservation due to differences in river and obstacle proximity.