In central Chile, increasing demand for water and decreasing runoff volumes due to drier conditions have placed catchments in this zone under water stress. However, scarcity of observed data records increases the difficulty of planning future water supply. Instrumental records suggest a reduction in streamflow over the last 56 years. However, this change is not statistically significant and the lack of meteorological stations with long records in this mountainous region hampers a deeper analysis, motivating the use of tree rings to analyse whether these changes are part of a long-term trend. This work represents the first high-elevation runoff reconstruction in Chile using 300 years of tree ring chronologies of Araucaria araucana and Astroceudrus chilensis. The upper part of Biobío river melting season runoff (October–March) and pluvial season runoff (April–September) was reconstructed and analysed to investigate the influence of large-scale climatic drivers on runoff generation, current drought trends and to improve the understanding of climate variability in this region. We obtained positive correlations between the 20-year moving average of reconstructed pluvial season runoff and reconstructed Pacific Decadal Oscillation (PDO), which is indicative of multi-decadal variability. We also found a negative correlation between the 11-year moving average of reconstructed melting season runoff and the PDO and positive correlations with the Southern Annular Mode (SAM). Important differences in the runoff variability of the upper and the lower part of the catchment were identified which are in part led by the influence of the large-scale climatic features that drive runoff generation in both regions. We found that the changes observed in the instrumental records are part of multi-decadal cycles led by the PDO and SAM for pluvial season runoff and melting season runoff, respectively.