Dissolved organic matter (DOM) is the most reactive pool of organic carbon in soil and one of the most important components of the global carbon cycle. Phototrophic biofilms growing at the soil−water interface in periodically flooding−drying soils like paddy fields consume and produce DOM during their growth and decomposition. However, the effects of phototrophic biofilms on DOM remain poorly understood in these settings. Here, we found that phototrophic biofilms transformed DOM similarly despite differences in soil types and initial DOM compositions, with stronger effects on DOM molecular composition than soil organic carbon and nutrient contents. Specifically, growth of phototrophic biofilms, especially those genera belonging to Proteobacteria and Cyanobacteria, increased the abundance of labile DOM compounds and richness of molecular formulae, while biofilm decomposition decreased the relative abundance of labile components. After a growth and decomposition cycle, phototrophic biofilms universally drove the accumulation of persistent DOM compounds in soil. Our results revealed how phototrophic biofilms shape the richness and changes in soil DOM at the molecular level and provide a reference for using phototrophic biofilms to increase DOM bioactivity and soil fertility in agricultural settings.