[Objective] This study investigates the synergistic and antagonistic effects of multiple factors on water self-purification capacity and examines self-purification efficiency of water under the combined influence of multiple factors, aiming to overcome the limitations of previous studies and provide new quantitative evidence for understanding water self-purification capacity in complex aquatic environments. [Methods] To investigate the interactions among multiple factors affecting water self-purification capacity, orthogonal experiments were conducted on water samples collected from the northwest side of a lake in Wuhan, which was affected by mixed pollution from domestic sewage and industrial wastewater and contained a microbial community. The orthogonal experiment results were analyzed using a combination of response surface methodology (RSM) and structural equation modeling (SEM) to reveal the nonlinear interaction mechanisms among typical influencing factors, including meteorological conditions, pollutant concentration background values, and biological activity. [Results] The results of orthogonal experiments showed that water self-purification efficiency (η), nitrification rate (r_N), and oxygen mass transfer efficiency (K_La) of water all varied nonlinearly across experimental groups, with maximum values observed at approximately temperature (T)=25 ℃, dissolved oxygen concentration (DO)=6 mg/L, flow rate (v)=0.1 m/s, microbial abundance (M)=5×10⁷ CFU/mL, and chemical oxygen demand (C)=100 mg/L. RSM analysis indicated that at the optimal parameter combination (T=24.8 ℃, DO=5.9 mg/L, v=0.27 m/s, C<100 mg/L, and M=4.7×10⁷ CFU/mL), η significantly increased to 83.24%, r_N exceeded 1 mg/(L·h), and K_La reached its maximum. In addition, in the binary factor interactions, the most significant interaction term was T×DO, with a synergistic contribution of 45.52%. SEM path analysis showed that v and T influenced the water self-purification process through both direct effect and indirect effect. Paths: v➝DO➝η; v➝DO➝Mi( microbial activity, including r_N and M)➝η; T➝DO➝η; T➝Mi➝η; T➝DO➝Mi➝η, where the direct effect of v on water self-purification process was 0.41, and the indirect effect was 0.25; and the direct effect of T on water self-purification process was 0.35, and the indirect effect was 0.25. The total effect of T and v on water self-purification process increased by 0.25 compared with the direct effect of each factor alone. [Conclusion] The results confirm that there is a synergistic amplification mechanism and significant threshold effects among different environmental influencing factors. Binary factor interactions not only show significant effects, but also factors originally negatively correlated with water self-purification capacity can significantly reduce their inhibitory influence on water self-purification efficiency after complex interactions with other factors. Additionally, factors such as flow rate and temperature affect the water self-purification process both directly and indirectly through their effects on other influencing factors. These findings provide a new quantitative basis for evaluating water self-purification ability and its controlling factors in complex aquatic environments.