Methylisothiazolinone (MIT) and Benzisothiazolinone (BIT) are two widely used non-oxidizing biocides of isothiazolinones. Their production and usage volume have sharply increased since the pandemic of COVID-19, inevitably leading to more release into water environment. However, their photochemical behaviors in water environment are still unclear. Therefore, this study investigated photodegradation properties of MIT and BIT in natural water under simulated sunlight. The results demonstrated that direct photolysis was mainly responsible for their photodegradation which occurred through their excited singlet states rather than triplet states. The quantum yields of MIT and BIT photodegradation were 11 - 13.6 × 10−4 and 2.43 - 5.79 × 10−4, respectively. pH had almost no effect on the photodegradation of MIT, while the photodegradation of BIT was significantly promoted under alkaline condition due to abundance of BIT in its deprotonated form (BIT-N−). Cl−, NO3− and dissolved organic matter (DOM) in natural water inhibited the photodegradation of both MIT and BIT, with the light screening effect of DOM being the most significantly inhibitory factor. The addition of other isothiazolinones, which possibly coexisted with MIT and BIT in actual condition, slightly inhibited the photodegradation of MIT and BIT. The estimated half-life under natural sunlight at a 30°N latitude was estimated to be approximately 1.1 days. The photodegradation pathways of MIT and BIT are similar, primarily initiated from the ring-opening at the N-S bond, with Frontier electron densities (FED) calculations suggesting the likelihood of oxidation and ·OH addition reactions at the O, N, and S sites. While the photodegradation products exhibited significantly reduced acute toxicity compared to their parent compounds, they nonetheless posed substantial chronic toxicity. These insights are vital for assessing the ecological impacts of MIT and BIT in aquatic environments.