Biotic interactions shape the community structure and function of ecosystems and thus play an important role in ecosystem management and restoration. To investigate how water temperature (related to the season) and water depth (related to spatial patterns of river morphology) affect macrophyte–bacterioplankton interactions in a groundwater-fed river, we conducted the structural equation modeling on datasets grouped by hydrological conditions. In addition to direct effects on macrophyte growth and/or bacterioplankton development, water temperature and water depth could both regulate the role of different nutrients (inorganic and organic) on affecting these biological indicators. Deeper water depth intensified the positive relationship between macrophytes and bacterioplankton, while higher temperature switched the relationship from being positive to negative. Our study provides empirical evidences that abiotic variables, even with relatively low fluctuations, play a critical role in regulating the patterns and strengths of interaction between macrophytes and bacterioplankton.