Aims: The aim of this study was to test the growth inhibition activity of isothiocyanates (ITCs), defence compounds of plants, against common human microbial pathogens. Methods and Results: In this study, we have tested the growth-inhibitory activity of a diverse collection of new and previously known representative ITCs of various structural classes against pathogenic bacteria, fungi and moulds by a serial dilution method. Generally, the compounds were more active against Gram-positive bacteria and fungi exhibiting species-specific bacteriostatic or bactericidal effect. The most active compounds inhibited the growth of both drug-susceptible and multi-drug-resistant (MDR) pathogens at micromolar concentrations. In the case of Mycobacterium tuberculosis, some compounds were more active against MDR, rather than against susceptible strains. The average antimicrobial activity for some of the new derivatives was significantly higher than that previously reported for the most active ITC compounds. The structure-activity relationship (SAR) established for various classes of ITC with Bacillus cereus (model organism for B.anthracis) followed a distinct pattern, thereby enabling prediction of new more efficient inhibitors. Remarkably, tested bacteria failed to develop resistance to ITC. While effectively inhibiting microbial growth, ITCs displayed moderate toxicity towards eukaryotic cells. Conclusions: High antimicrobial activity coupled with moderate toxicity grants further thorough studies of the ITC compounds aimed at elucidation of their cellular targets and inhibitory mechanism. Significance and Impact of the Study: This systematic study identified new ITC compounds highly active against common human microbial pathogens at the concentrations comparable with those for currently used antimicrobial drugs (e.g. rifampicin and fluconazole). Tested representative pathogens do not develop resistance to the inhibitors. These properties justify further evaluation of ITC compounds as potential antimicrobial agents for medicinal use and for industrial applications.