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The effects of nisin and monolaurin, alone and in combination, were investigated on Bacillus licheniformis spores in milk at 37 °C. In the absence of inhibitors, germinated spores developed into growing vegetative cells and started sporulation at the end of the exponential phase. In the presence of nisin (25 IU ml−1), spore outgrowth was inhibited (4 log10 reduction at 10 h). Regrowth appeared between 10 and 24 h and reached a high population level (1·25 × 108 cfu ml−1) after 7 d. Monolaurin (250 μg ml−1) had a bacteriostatic effect during the first 10 h but thereafter, regrowth occurred slowly with a population level after 7 d (4 × 105 cfu ml−1) lower than that of nisin. Different combined effects of nisin (between 0 and 42 IU ml−1), monolaurin (ranging from 0 to 300 μg ml−1), pH values (between 5·0 and 7·0) and spore loads (103, 104, 105 spores ml−1) were investigated using a Doehlert matrix in order to study the main effects of these factors and the different interactions. Results were analysed using the Response Surface Methodology (RSM) and indicated that nisin and monolaurin had no action on spores before germination; only pH values had a significant effect (P ≤ 0·001), i.e. spore count decreased as the pH value increased in relation to germination. Sublethal concentrations of nisin (30 IU ml−1) and monolaurin (100 μg ml−1) in combination acted synergistically on outgrown spores and vegetative cells, showing total inhibition at pH 6·0, without regrowth, within 7 d at 37 °C.