Population growth and economic development are driving changes in land use/land cover (LULC) of the transboundary Lower Mekong River Basin (LMB), posing a serious threat to the integrity of the river system. Using data collected on a monthly basis over 30 years (1985–2015) at 14 stations located along the Lower Mekong river, this study explores whether spatiotemporal relationships exist between LULC changes and instream concentrations of total suspended solids (TSS) and nitrate—as proxies of water quality. The results show seasonal influences where temporal patterns of instream TSS and nitrate concentrations mirror patterns detected for discharge. Changes in LULC influenced instream TSS and nitrate levels differently over time and space. The seasonal Mann–Kendall (SMK) confirmed significant reduction of instream TSS concentrations at six stations (p < 0.05), while nitrate levels increased at five stations (p < 0.05), predominantly in stations located in the upper section of the basin where forest areas and mountainous topography dominate the landscape. Temporal correlation analyses point to the conversion of grassland (r = −0.61, p < 0.01) to paddy fields (r = 0.63, p < 0.01) and urban areas (r = 0.44, p < 0.05) as the changes in LULC that mostly impact instream nitrate contents. The reduction of TSS appears influenced by increased forest land cover (r = −0.72, p < 0.01) and by the development and operation of hydropower projects in the upper Mekong River. Spatial correlation analyses showed positive associations between forest land cover and instream concentrations of TSS (r = 0.64, p = 0.01) and nitrate (r = 0.54, p < 0.05), indicating that this type of LULC was heavily disturbed and harvested, resulting in soil erosion and runoff of nitrate rich sediment during the Wet season. Our results show that enhanced understanding of how LULC changes influence instream water quality at spatial and temporal scales is vital for assessing potential impacts of future land and water resource development on freshwater resources of the LMB.