Veterinary　antibiotics　and　herbicides　could　be　simultaneously　ingested　by　livestock　(especially　cattle)　due　to　their　co-occurrence　in　feed,　disrupting　redox　homeostasis.　However,　limited　information　is　available　on　the　combined　molecular-level　effects　of　these　agrochemicals　on　antioxidant　enzymes.　In　this　study,　sulfadiazine　(SD)　and　metamitron　(MET)　were　respectively　selected　as　the　representatives　of　veterinary　antibiotics　and　herbicides　to　explore　their　combined　toxic　effects　on　SOD　and　the　underlying　molecular　mechanisms,　using　multi-spectroscopic　technologies,　X-ray　photoelectron　spectroscopy　(XPS),　and　molecular　docking.　Binding　and　conformation　characterizations　revealed　that,　at　specific　compound-to-enzyme　ratios,　SOD　inactivation　predominantly　originated　from　direct　binding　of　test　chemicals　at　key　sites　rather　than　compound-induced　conformational　changes.　Furthermore,　fluorescence　quenching　analysis　demonstrated　1:1　binding　stoichiometry,　indicating　the　only　one　preferential　binding　site　for　SD　and　MET　in　SOD.　An　integrated　‘interaction-microenvironment-conformation’　evaluation　framework　was　established　to　judge　the　joint　toxic　effects　between　SD　and　MET,　suggesting　that　these　chemicals　competitively　interacted　with　the　same　binding　site　to　antagonistically　inhibit　SOD　activity.　Considering　the　proximal　amino　acid　residues　(such　as　glutamate,　arginine,　and　tyrosine　residues)　confirmed　by　activity　test,　synchronous　fluorescence,　and　XPS,　a　key　binding　pocket　in　the　SOD　inter-subunit　cavity　was　identified　as　the　interaction　site　for　both　SD　and　MET.　Computational　simulations　further　validated　and　visualized　the　binding　characteristics　of　test　chemicals　within　this　key　SOD　dimer　interface.　This　study　elucidates　the　interaction　characteristics　between　SOD　and　typical　agrochemicals,　providing　a　new　insight　and　methodology　for　assessing　the　molecular-level　health　risks　of　mixed　agrochemicals.　©　2025　Elsevier　B.V.