BACKGROUNDUtilizing fungicides to protect crops from diseases is an effective method, and novel eco‐friendly plant‐derived fungicides with high efficiency and low toxicity are urgent requirements for sustainable crop protection.RESULTTwo series of rosin‐based fungicides (totally 35) were designed and synthesized. In vitro fungicidal activity revealed that Compound 6a (Co. 6a) effectively inhibited the growth of Valsa mali [median effective concentration (EC50) = 0.627 μg mL−1], and in vivo fungicidal activity suggested a significant protective efficacy of Co. 6a in protecting both apple branches (35.12% to 75.20%) and apples (75.86% to 90.82%). Quantum chemical calculations (via density functional theory) results indicated that the primary active site of Co. 6a lies in its amide structure. Mycelial morphology and physiology were investigated to elucidate the mode‐of‐action of Co. 6a, and suggested that Co. 6a produced significant cell membrane damage, accelerated electrolyte leakage, decreased succinate dehydrogenase (SDH) protein activity, and impaired physiological and biochemical functions, culminating in mycelial mortality. Molecular docking analysis revealed a robust binding energy (ΔE = −7.29 kcal mol−1) between Co. 6a and SDH. Subsequently, biosafety evaluations confirmed the environmentally‐friendly nature of Co. 6a via the zebrafish model, yet toxicological results indicated that Co. 6a at median lethal concentration [LC50(96)] damaged the gills, liver and intestines of zebrafish.CONCLUSIONThe above research offers a theoretical foundation for exploiting eco‐friendly rosin‐based fungicidal candidates in sustainable crop protection. © 2024 Society of Chemical Industry.