Despite numerous papers being published on the use of hurdle technology to control food-borne pathogens or spoilage organisms, there is no commonly accepted methodology to quantify the level of synergistic activity. This paper describes a method to quantify in vitro the synergistic activity of antibacterial agents against bacteria. Initially, a microtiter plate growth assay was used to determine the inhibitory concentrations of four "natural" antimicrobials (nisin, lauricidin, totarol, and the lactoperoxidase system (LPS)) against a panel of eight bacteria. Using the same microtiter system, the impact of various combinations of antimicrobials was assessed. The degree of synergy was based on the analysis of three criteria: (1) increase in lag phase, (2) reduction in culture density after 24 h, (3) and residual viability at 24 h. Only the lactoperoxidase system was active against all the Gram-positive and Gram-negative bacteria tested. Nisin, lauricidin, and totarol were only effective against the Gram-positive bacteria. The method successfully identified three combinations (nisin-lauricidin, LPS-nisin, and LPS-lauricidin) previously reported to have synergistic activity and highlighted the synergistic activity of two novel combinations (nisin-totarol and LPS-totarol). The development of a quick and reliable method to identify and quantify synergistic activity is a useful screening tool to establish preservative techniques that could have potential antimicrobial synergy in food-based systems.