Alicyclobacillus spp. has recently received much attention due to its implication in the spoilage of pasteurized fruit juices, which was characterized by the production of a smoky, medicinal and antiseptic off-odor attributed to guaiacol. The species those are known to produce guaiacol are A. acidoterrestris, A. acidiphilus and A. herbarius. The effect of ε-polylysine on growth and guaicol production of these three species of Alicyclobacillus spp. was evaluated. Minimal inhibitory concentration (MIC) of ε-polylysine was determined in AAM medium by using broth dilution method. The sensitivity of Alicyclobacillus spp. to ε-polylysine was species-dependent, and MIC values ranged from 1.25 mg/mL to 5 mg/mL against vegetative cells and from 2.5 mg/mL to 5 mg/mL against the spores. Then, various concentrations of ε-polylysine were added to the model apple juice. When the dose of ε-polylysine increased to 4 MIC, Alicyclobacillus counts were reduced below 2.66 log colony forming unit (CFU)/mL, and no guaiacol was detected after 7 d incubation. The results can be very useful for better control of Alicyclobacillus-related spoilage in the fruit juice and beverage industry.