The water quality of the Mun River, one of the largest tributaries of the Mekong River and an important agricultural area in Thailand, is investigated to determine its status, identify spatiotemporal variations and distinguish the potential causes. Water quality dataset based on monitoring in the last two decades (1997–2017) from 21 monitoring sites distributed across the basin were analyzed using seasonal Kendall test and water quality index (WQI) method. The Kendall test shows significant declines in fecal coliform bacteria (FCB) and ammonia (NH3) in the upper reaches and increases in nitrate (NO3) and NH3 in the lower reaches. Strong temporal and spatial fluctuations were observed in both the concentrations of individual parameters and the WQI values. Seasonal variation of water quality was observed at each monitoring site. WQI values in August (flood season) were generally among the lowest, compared to other seasons. Spatially, sites in the upper reaches generally having lower WQI values than those in the lower reaches. Excessive phosphorus is the primary cause of water quality degradation in the upper reaches, while nitrogen is the primary parameter for water quality degradation in the lower reaches. Urban built-up land is an important “source” of water pollutants in the lower basin, while agricultural land plays a dual role, affecting across the basin.