In this study, new Tetracaine thiosemicarbazide derivatives were designed, synthesized, and evaluated for their antimicrobial activity. These compounds (2a-l) were characterized using spectroscopic (H-1-C-13 NMR, FT-IR and HR-MS) methods and tested for their antimicrobial activity against various Gram-positive and Gram-negative bacterial strains (Staphylococcus aureus ATCC 29213: Enterococcus faecium ATCC 29212; Escherichia coli ATCC 25922; Pseudomonas aeruginosa ATCC 27853), and fungus (Candida albicans ATCC 10231). The microbroth dilution method was utilized for the determination of the minimum inhibitory concentration (MIC) values of novel molecules. The antimicrobial activities of the molecules were found to have a wide range of MIC values with 4-256 mu g/mL. Among the synthesized molecules, compound 2 h (2-[4-(butylamine)benzoyl]-N-(3,5-bistrifluoromethylphenyl)hydrazine-1-carbothioamide) showed the best MIC value for 4 (mu g/mL) to S. aeurus isolate and 8 (mu g/mL) to C. albicans, respectively. Compounds 2e (2-[4-(butylamine)benzoyl]-N-(2-fluorophenyl)hydrazine-1-carbothioamide) and 2f (2-[4-(butylamine)benzoyl]-N-(4-nitrophenyl)hydrazine-1-carbothioamide) showed moderate activity to C. albicans and S. aeurus with a 16 (mu g/mL) MIC value.