Microbial growth and inactivation kinetics in food can be predicted when the effects of food and environmental conditions on microbial responses are known. However, the prediction result might not be accurate due to microbial variability. To ensure food safety and quality, knowledge on the sources of variability and the magnitude is needed to prioritize their importance. Listeria monocytogenes and Lactobacillus plantarum were selected as model organisms to represent pathogenic and spoilage microorganisms. The result of this project underlines that many variability factors are important, but some are more important than others. Depending on the process characteristics, microbiological variability, especially strain variability, and in particular for thermal inactivation, will be the most determining factor affecting the final contamination level. This strain variability, however, is inherent to living organisms and challenges food processors because strain variability cannot be well controlled unless complete inactivation is realized and no recontamination occurs within the food production chain. The integration of strain variability in prediction of microbial kinetics is, therefore, required in quantitative microbiology to obtain a more realistic prediction.