It is well documented that climate change is growing the occurrence of droughts in many regions that may affect water resources through their water quality and quantity. Nevertheless, in Chile the information is scarce about how water quality of high flow rivers response to droughts. This paper seeks to investigate the effect of an unprecedented mega-drought (2010-2015) on the water quality of Biobío river (36°45′ – 38°49′S and 71º00′ – 73º20′W) Central Chile, one of the largest Chilean rivers that provides abundant freshwater for human consumption, irrigation and hydropower. The water quality (surface water temperature, pH, dissolved oxygen, electrical conductivity, BOD5, suspended solids major ions such as chloride, sodium, nutrients and heavy metals), during the mega-drought (2010-2015) was compared with the water quality from a non-drought period (2000-2009) in two reaches (upstream and downstream) of the river, and we investigated differences in physicochemical parameters in both reaches. Mann-Kendall and seasonal Mann-Kendall trend analyses and Theil-Sen estimator were used to investigate trends of the reaches. Results show during the mega-drought a slightly decrease trend in pH, nutrients (NO2-, NH4+, NO3-, Total P) and suspended solids, and an increase trend in surface water temperature and trace metals (Cr, Fe and Co) in both reaches, mainly during summer, although in winter were also changes found. During the mega-drought, upstream variability suggested more constant concentrations in nutrients and metals than downstream. Based on calculated slopes of major ions and metals trends, during summer seasons more rapid decreasing trends were present than in winters, mainly downstream. These results suggest that seasonal changes in surface water temperature, discharge and precipitation may have some negative changes on water quality content. These highlight that seasonal changes and a progressive reduction of river flow availability affecting the vulnerability of the water quality in this key Chilean river. The outcomes from this research are expected to support and influence actions for policy making to address water quality goals in watershed river management. Hence, to better guide future management and take into account the effects of a river discharge reduction under low flow conditions as a consequence of more frequent drought periods.