Aims: Compare the survival behaviour of food spoilage micro-organisms treated with sequential doses or all at once treatments of eugenol. Methods and Results: Staphylococcus carnosus, Listeria innocua, Escherichia coli and Pseudomonas fluorescens were exposed to a minimal lethal concentration (MLC) initially, or to half doses over time, with first half dose applied immediately and a second half dose applied after 3, 4, 6 and 8 h, of eugenol. Direct plate counts were determined at regular time intervals. Population dynamics were analysed using a combined growth and mortality model. Effect of sequential dosing varied significantly between tested organisms. High antimicrobial efficacy on E. coli K12 was observed regardless of timing of the two doses. Reduced effectiveness was observed against Staph. carnosus and L. innocua the later the second half dose was applied. Complete cell reduction occurred after immediate exposure to full MLC dose, while sequential half doses were bacteriostatic regardless of application times. Conclusion: Time in between antimicrobial dose application had substantial impact on effectiveness, attributed to organisms becoming more tolerant. Significance and Impact of the Study: This study contributes to the evaluation of encapsulated antimicrobial systems, where antimicrobials are released over time and antimicrobial concentrations may only reach MLC levels after some time.