The increased consumer awareness of health-associated risks of synthetic preservatives along with increasing antimicrobial resistance drive research towards exploring new natural antimicrobial compounds. The Brassicaceae plant family is a potential source of new antimicrobials, e.g. as food preservatives. These plants abundantly produce glucosinolates (GSLs), which serve as precursors of antimicrobial isothiocyanates (ITCs), formed upon contact of GSLs and myrosinase. GSLs as well as ITCs are structurally diverse, differing in side chain configuration. The main aim of this thesis research was to explore the potential of ITCs as natural antimicrobials. For this, the formation, the analysis, the reactivity, and the (quantitative) structure-antimicrobial activity relationships ((Q)SAR) of ITCs were studied. A simultaneous RP-UHPLC-ESI-MSn analysis of various GSLs and ITCs was developed and can be useful to monitor the in vitro enzymatic degradation of GSLs to ITCs in complex mixtures. Furthermore, this study confirms that ITCs from subclasses x-(methylsulfinyl)alkyl and x-(methylsulfonyl)alkyl had potent antimicrobial activity and, therefore, might be potential new natural food preservatives, but their reactivity with food matrix components should be considered. Furthermore, the QSAR models developed in this thesis can be applied to predict antibacterial activity of new ITCs and (natural) mixtures of ITCs. Overall, ITCs are promising natural antimicrobial candidates worth further studies.