The Santiago River (Jalisco) is a major waterway in western Mexico and has received considerable attention due to its severe pollution. Understanding the impact of reduced human activity on water quality in the Santiago River during the COVID-19 lockdown (April-May 2020) is critical for river management and restoration. However, there has been no published study in this context, presenting a significant knowledge gap. Hence, this study focuses on determining if the nationwide COVID-19 lockdown influenced or improved surface water quality in a 262-km stretch of the Santiago River upstream. Data for 15 water quality parameters collected during the lockdown were compared to levels obtained in 2019 (pre-lockdown), 2021 (unlock), and the previous eleven years (2009-2019). The values of turbidity, BOD, COD, TSS, f. coli, t. coli, nitrate, sulfate, and Pb decreased by 4-36%, while pH, EC, total nitrogen, and As increased by 0.3-21% during the lockdown compared to the pre-lockdown period, indicating a reduction in organic load in the river due to the temporary closure of industrial and commercial activities. An eleven-year comparison estimated a 0-38% decline in pH, TSS, COD, total nitrogen, sulfates, nitrates, and Pb. The unlock-period comparison showed a significant rise of 3-37% in all parameters except As, highlighting the potential repercussions of restoring activity along the Santiago River. Estimated water quality indices demonstrated short-term improvements in river water quality during the lockdown when compared to other time periods investigated. According to factor analysis, the main pollution sources influencing river water quality were untreated household sewage, industrial wastewater, and agricultural effluents. Overall, our analysis showed that the COVID-19-imposed lockdown improved the water quality of the Santiago River, laying the groundwork for local officials to identify pollution sources and better support environmental policies and water quality improvement plans. Supplementary Information: The online version contains supplementary material available at 10.1007/s12665-022-10430-9.