This study reports the synthesis and analysis of biologically active acylthiourea compounds (1 and 2) with a cyclohexyl moiety. The compounds were characterized using UV-Visible, FT-IR, 1H/13C NMR, and elemental analysis. The crystal structure of 2 was solved, revealing intra- and inter-molecular hydrogen bonds. Density functional theory (DFT) calculations provided insights into chemical reactivity and non-covalent interactions. Cytotoxicity assays showed the cyclohexyl group enhanced the activity of compound 2 compared to compound 1. Epoxide hydrolase 1 was predicted as the enzyme target for both compounds. We modeled the structure of epoxide hydrolase 1 and performed molecular dynamics simulation and docking studies. Additionally, in silico docking with SARS-CoV-2 main protease, human ACE2, and avian influenza H5N1 hemagglutinin indicated strong binding potential of the compounds. This integrated approach improves our understanding of the biological potential of acylthiourea derivatives. C1 [Haribabu, Jebiti; Echeverria, Cesar] Univ Atacama, Fac Med, ATACAMA OMICS, Carreras 1579, Copiapo 1532502, Chile. [Haribabu, Jebiti; Swaminathan, Srividya] Univ Madras, Ctr Adv Study Crystallog & Biophys, Guindy Campus, Chennai 600025, Tamil Nadu, India. [Madhavan, Geetha; Dasararaju, Gayathri] Chennai Inst Technol, Chennai 600069, Tamil Nadu, India. [Panneerselvam, Murugesan] Univ Fed Fluminense, MolMod CS, Inst Quim, Campos Valonginho S-N, BR-2402014 Niteroi, RJ, Brazil. [Moraga, Daniel] Univ Tarapaca, Dept Ciencias Biomed, Lab Fisiol, Fac Med, Arica 1000000, Chile. [Arulraj, Arunachalam] Univ Tecnol Metropolitana UTEM, Fac Ingn, Dept Elect, Jose Pedro Alessandri 1242, Santiago 7800002, Chile. [Mangalaraja, Ramalinga Viswanathan] Univ Adolfo Ibanez, Fac Sci & Engn, Diagonal Torres 2640, Santiago, Chile. [Mangalaraja, Ramalinga Viswanathan] Karpagam Acad Higher Educ, Fac Engn, Dept Mech Engn, Coimbatore 641021, Tamil Nadu, India. [Kokkarachedu, Varaprasad] Univ San Sebastian, Fac Ingn Arquitectura & Diseno, Lientur 1457, Concepcion, Chile. [Santibanez, Juan F.] Univ Belgrade, Inst Med Res, Natl Inst Republ Serbia, Belgrade, Serbia. [Santibanez, Juan F.] Univ Bernardo OHiggins, Integrat Ctr Biol & Appl Chem CIBQA, Santiago, Chile. [Ramirez-Tagle, Rodrigo] Univ Cent Chile, Fac Ingn & Arquitectura, Santiago, Chile. C3 Universidad de Atacama; University of Madras; Chennai Institute of Technology; Universidade Federal Fluminense; Universidad de Tarapaca; Universidad Tecnologica Metropolitana; Universidad Adolfo Ibanez; Karpagam Academy of Higher Education (KAHE); Universidad San Sebastian; University of Belgrade; Universidad Bernardo O'Higgins; Universidad Central de Chile