Viruses saturate environments throughout the world and play key roles in microbial food webs, yet how viral activities affect dissolved organic matter (DOM) processing in natural environments remains elusive. We established a large-scale long-term macrocosm experiment to explore viral dynamics and their potential impacts on microbial mortality and DOM quantity and quality in starved and stratified ecosystems. High viral infection dynamics and the virus-induced cell lysis (6.23–64.68% d−1) was found in the starved seawater macrocosm, which contributed to a significant transformation of microbial biomass into DOM (0.72–5.32 μg L−1 d−1). In the stratified macrocosm, a substantial amount of viral lysate DOM (2.43–17.87 μg L−1 d−1) was released into the upper riverine water, and viral lysis and DOM release (0.35–5.75 μg L−1 d−1) were reduced in the mixed water layer between riverine water and seawater. Viral lysis was stimulated at the bottom of stratified macrocosm, potentially fueled by the sinking of particulate organic carbon. Significant positive and negative associations between lytic viral production and different fluorescent DOM components were found in the starved and stratified macrocosm, indicating the potentially complex viral impacts on the production and utilization of DOM. Results also revealed the significant viral contribution to pools of both relatively higher molecular weight labile DOM and lower molecular weight recalcitrant DOM. Our study suggests that viruses have heterogeneous impact on the cycling and fate of DOM in aquatic environments.