The agar diffusion bioassay is the most widely used method for the quantification of nisin, due to its high sensitivity, simplicity, and cost-effectiveness. This method is based on the measurement of the inhibition zone produced in nisin-sensitive microorganisms. The size of the zone is affected by many factors, such as nisin-sensitive strain, amount of added agar and surfactant, and pre-diffusion step. This research aims to evaluate the effects of nisin-sensitive strains and pre-diffusion on the accuracy and precision of nisin quantification. Three strains of nisin-sensitive microorganisms (Micrococcus luteus, Lactobacillus sakei, Brochothrix thermosphacta) were tested along with three different incubation processes. The best combination was the method using L. sakei as an indicator strain with pre-diffusion at 4degreesC for 24 h. Compared with M. luteus and B. thermosphacta, L. sakei gave more accurate and reproducible results. Moreover, the pre-diffusion step resulted in larger inhibition zones and more precise results. Finally, the best combination was validated and compared with the method that is usually used and the result showed that the method using L. sakei with pre-diffusion gave more accurate and precise results.