The Tortonian–Messinian transition is associated with important climatic and oceanographic changes in the Mediterranean Basin, which have shaped both the biotic and abiotic nature of this setting. The morphological variability of the planktonic foraminifera Globorotalia menardii, a species that is highly sensitive to water column structure, has been investigated from the sedimentary archive of three Cretan sections across a west–east transect covering the Tortonian–Messinian Boundary. The present work explicitly focuses on test-size and coiling direction changes occurring during the 7.36–7.24 Ma time slice. On such a short timescale, the most important morphological differentiation accounts for the average size of G. menardii, which is mostly associated with evolutionary adaptation to new ecological niches during the latest Tortonian as a response to the environmental perturbations and ecological stress conditions preceding the Tortonian–Messinian Boundary. A combined thermal and/or salinity-driven stratification and thermocline development hypothesis has been suggested to explain the observed size variability. To ameliorate the accuracy of the proposed model and further determine which environmental parameter reflects the optimum conditions of the analysed species, additional sea surface temperature and salinity data derived from the same sampling intervals of the studied or additional Mediterranean sites are needed. The coiling direction of this species within the study time interval remained constant and not environmentally controlled.