The infectious diseases caused by antimicrobials pathogens are difficult, harmful, and sometimes impossible to treat. Therefore, design new drugs to treat antimicrobial infections is the biggest challenge for modern medicine. 2-Thioxo-4-imidazolidinone is used for the synthesis of a wide variety of new substituted imidazolidinone derivatives. In this study, we designing eight novel compounds derived for the 2-thioxo-4-imidazolidinones (5a-h). The preparation was in two steps via Micheal addition of phenyl hydrazide (2a), 4-methyl phenyl hydrazide 2b on N-substituted maleimides 1a-d in ethanol, and the second step by reaction of maleimide derivatives with cyclohexyl isothiocyanate 4 in acetonitrile. The chemical structures of the compounds were identified using FT-IR, 1H-NMR, 13C-NMR, and mass spectra, as well as the melting point. The antibacterial and antifungal evaluation was carried out to target their activities. Compound N-(5-(2-((4-chlorophenyl)amino)-2-oxoethyl)-3-cyclohexyl-4-oxo-2-thioxoimidazolidin-1-yl) benzamide (5b) exhibited antibacterial activity toward Staphylococcus aureus and Pseudomonas aeruginosa with equal minimum inhibitory concentration (MIC) values of 25 mg/mL. Compounds N-(3-cyclohexyl-4-oxo-5-(2-oxo-2-(phenylamino)ethyl)-2-thioxoimidazolidin-1-yl) benzamide (5a), N-(5-(2-((4-chlorophenyl)amino)-2-oxoethyl)-3-cyclohexyl-4-oxo-2-thioxoimidazolidin-1-yl) benzamide (5b), N-(5-(2-((4-bromophenyl)amino)-2-oxoethyl)-3-cyclohexyl-4-oxo-2-thioxo imidazolidin-1-yl) benzamide (5c), and N-(3-cyclohexyl-4-oxo-5-(2-oxo-2-(phenylamino)ethyl)-2-thioxoimidazolidin-1-yl)-4-methyl benzamide (5e) exhibited antifungal activity toward Candida albicans, while all compounds exhibited antifungal activity toward Aspergillus niger except for compound 5h, with various MIC values. In conclusion, the results demonstrate that the new compounds have to promise as antifungal agents. Moreover, compound 5b could develop as an antibacterial agent.