Upper Cretaceous-Cenozoic marine sequences preserved between 30 and 350 masl across southern South Africa record a complex history of climatic and tectonic changes. In this study, we measure the strontium (Sr) isotope composition of fossil shark teeth, echinoderms, corals and oyster shells to chronostratigraphically constrain the ages of these sequences. The method requires careful petrographic screening and micro-drilling of the samples to avoid possible alteration by diagenesis. To assess palaeoenvironmental effects in the shells we measured the Mg/Ca elemental ratios and O isotope values using electron microprobe analysis (EMPA) and secondary ion mass spectrometry (SIMS). In addition, we employed carbonate clumped isotope thermometry to test palaeotemperatures reconstruction. The analysis of recent to modern stromatolites by clumped isotopes yields an average temperature of 20.2°C, in agreement with present day observations. The fossil oyster shells suggest a warmer (23.0°C) seawater palaeotemperature, possibly due to major deglaciation and sea-level rise during the Neogene. We also find that transgressions occurred above 200 to 350 m elevation during: 1) the Campanian-Maastrichian (~75 Ma); 2) the mid-Oligocene (32 to 26 Ma); and 3) the Messinian-Zanclean (6 to 5 Ma). These three episodes are linked to well-known variations in global sea level and regional tectonic processes that could have affected the continental margin differently. The most recent transgression coincides with a maximum global sea-level rise of ~50 m at ca. 5.3 Ma, and a worldwide plate kinematic change around 6 Ma, which in Eurasia led to the closure of the Mediterranean Sea. In the Eastern Cape of South Africa, the new dates of analyzed oyster shells constrain a minimum uplift rate of ca. 150 m/Myr during this tectonic activity. The results have important implications for robust calibration of relative sea level in southern Africa.