Redox transformation of mercury (Hg) is critical for Hg exchange at the air-water interface. However, the superoxide radicals (O2•─) contribution of microalgal-fungal symbiotic systems in lake water to Hg(II) reduction is mainly unknown. Here, we studied the enhanced potential for O2•─ production by the coupling effect between microalgae and fungi. The relationships between microenvironment, microorganisms, and O2•─ production were also investigated. Furthermore, the implication of O2•─ for Hg(II) reduction was explored. The results showed that the coupling effect of microalgae and fungi enhanced O2•─ generation in the symbiotic systems, and the O2•─ generation peaked on day 4 in the lake water at 160.51±13.06~173.28±18.21 μmol/kg FW (fresh weight). In addition, O2•− exhibited circadian fluctuations that correlated with changes in dissolved oxygen content and redox potential on the inter-spherical interface of microalgal-fungal consortia. Partial least squares path modeling (PLS-PM) indicates that O2•─ formation was primarily associated with microenvironmental factors and microbial metabolic processes. The experimental results suggest that O2•─ in the microalgal-fungal systems could mediate Hg(II) reduction, promoting Hg conversion and cycling. The findings highlight the importance of microalgae and fungal symbiotic systems in Hg transformation in aquatic environments. Environmental ImplicationThe redox cycling between elemental mercury [Hg(0)] and mercuric species [Hg(II)] is a crucial process influencing the fate of Hg in natural ecosystems. While the reduction of Hg(II) by superoxide radicals (O2•−) has been documented, the role of O2•−-mediated Hg(II) reduction in microalgal-fungal symbiotic systems remains unclear. This study investigated enhanced O2•− production due to microalgal-fungal synergy and its mechanisms. The contribution of O2•− to Hg(II) reduction was also assessed. These findings highlighted the previously unrecognized high levels of O2•− production in microalgal-fungal symbiotic systems, which could significantly impact the biogeochemical cycling of Hg.