β-blockers are known to have negative effects on fish and other aquatic animal species, so their removal is key for preserving aquatic ecosystems. To reduce the risks related to β-blockers, it is necessary to assess their effects and develop more effective treatments such as advanced oxidation processes. Improving sewage treatments is a critical approach to reducing β-blockers in aquatic environments. In this work, for the first time, the direct and indirect photolysis of nadolol (NAD) was investigated under different light sources (simulated solar (SS), UV-LED, and UV radiations) in ultrapure water. Indirect photolysis by H 2 O 2 showed 1.5, 2.1, and 5.6 times higher NAD degradation efficiency than direct photolysis under mentioned irradiations. This effect was particularly pronounced in the presence of UV radiation, in which the degradation efficiency of NAD was the highest (80.2%). Computational analysis based on density functional theory calculations, together with the results of NAD photodegradation efficiency in the presence of radical scavengers (isopropanol and benzoquinone), was used to propose the NAD degradation mechanism. Sixteen degradation intermediates were proposed, along with their NMR chemical shifts. Also, this study analyzed the degree of catalase activity, lipid peroxidation, and hydroxyl radicals neutralization of NAD and its photodegradation mixtures obtained after indirect photolysis. The degree of mineralization and in vitro toxicity of NAD and its degradation intermediates obtained in the presence of UV/H 2 O 2 were assessed.