The Knoevenagel reaction is an essential synthetic tool in the organic and medicinal chemistry of thiazolidin-4-one derivatives. In the present work, the application of ethylenediamine diacetate (EDDA) as an effective catalyst for the interaction of 2-thioxothiazolidin-4-one with 4-(tert-butyl)cyclohexanone is proposed. The structure of novel synthesized 5-[4-(tert-butyl)cyclohexylidene]-2-thioxothiazolidin-4-one (yield 61%) was confirmed by¹H-,¹³C-NMR, LC-MS, IR, and UV spectra. Drug-like properties of the synthesized compound were evaluated in silico using the SwissAdme, and their potential antimicrobial activity against 15 strains of Gram-positive and Gram-negative bacteria as well as yeasts was evaluated in vitro. The synthesized compound possesses satisfactory drug-like parameters and promising antimicrobial properties and presents interest as a prospective intermediate for the forthcoming design of biologically active small molecules. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.