Compounds bearing the 1,4-naphthoquinone ring and sulphur containing derivatives had been the subject of much interest due to their antimicrobial, antiviral, anti-inflammatory and anti-tumoural activities. Design of new derivatives of naphthoquinones, in order to improve their pharmaceutical properties, is still a research area of utmost importance. In the present study, we describe the synthesis, physico-chemical properties and evaluation of antimicrobial activity of new 2-mercapto-3-substituted-1,4-naphthoquinones. For the synthesis we used different 2-chloro-3-substituted-1,4-naphthoquinones and thiourea. The new naphthoquinone derivatives were characterized by elemental analysis, UV-VIS and IR spectrometry, mass spectrometry and H-1-NMR. All this methods confirmed the structure of the compounds. The antimicrobial activity was tested on several Gram-positive and Gram-negative bacteria using disk diffusion technique. In general, the compounds were active against tested Gram-positive bacteria. This encourages further studies for application of these compounds as antibiotic therapy.