During the middle and late Holocene, the Iberian Peninsula underwent large climatic and hydrologic changes, but the temporal resolution and regional distribution of available palaeoenvironmental records is still insufficient for a comprehensive assessment of the regional variability. The high sedimentation rate in karstic, meromictic Montcortès Lake (Catalan pre-Pyrenees) allows for a detailed reconstruction of the regional palaeoecology over the last 5,340 years using diatom analysis, aquatic pollen, sedimentological data, and historic documentary records. Results show marked fluctuations in diatom species assemblage composition, mainly between dominant Cyclotella taxa and small Fragilariales. We suggest that the conspicuous alternation between Cyclotella comta and C. cyclopuncta reflects changes in trophic state, while the succession of centric and pennate species most likely reflects changes in the hydrology of the lake. The diatom assemblages were used to identify six main phases: (1) high productivity and likely lower lake levels before 2350 BC, (2) lower lake levels and a strong arid phase between 2350 and 1850 BC, (3) lake level increase between 1850 and 850 BC, (4) relatively high lake level with fluctuating conditions during the Iberian and Roman Epochs (650 BC–350 AD), (5) lower lake levels, unfavourable conditions for diatom preservation, eutrophication and erosion triggered by increased human activities in the watershed during the Medieval Climate Anomaly (900–1300 AD), and (6) relatively higher lake levels during the LIA (1380–1850 AD) and afterwards. The combined study of diatoms, algae and pollen provides a detailed reconstruction of past climate, which refines understanding of regional environmental variability and interactions between climate and socio-economic conditions in the Pyrenees.