Aims To investigate the effect of different well sizes and pre-diffusion times at 4 degrees C, on the sensitivity, accuracy and precision of nisin quantification by agar diffusion bioassay. Methods and results Nisin solution (0 center dot 625125gml1) was filled in wells (3 center dot 5mm or 7mm diameter) made on agar plates inoculated with Micrococcus luteus, followed by pre-diffusion (0, 24, 48 or 72h), incubation and measurement of inhibition zone. Regression analysis indicated that wells with 3 center dot 5mm diameter had smaller standard deviation and higher predictive accuracy, compared to wells with 7mm diameter. Based on Tukey's test, pre-diffusion resulted in significantly different inhibition zones at different nisin concentrations. Pre-diffusion also improved sensitivity of the assay. Different regression models were considered to explore the relationship between inhibition zone and nisin concentration for different pre-diffusion times. A spline model was determined to be the best-fit model, and 48h was the best pre-diffusion time. Conclusions Wells with 3 center dot 5mm diameter demonstrated higher accuracy for nisin quantification compared to wells with 7mm diameter. 48h was the best pre-diffusion time for nisin concentration in the range 0 center dot 625125gml1. Significance and impact of the study The findings from this study will be helpful in quantifying nisin and compounds with antimicrobial properties accurately over a wide range of concentrations using agar diffusion bioassay.