Food waste caused by microbial spoilage poses negative economic and environmental impact at a global level. Alicyclobacillus acidoterrestris is a spore-forming, thermophilic bacterium that can cause spoilage in shelf-stable apple juice by producing off-odor compounds, including guaiacol. This study investigated the efficacies of natural glycolipid (NG), a novel antimicrobial, extracted from fungal fermentation, in controlling vegetative cells and spores of A. acidoterrestris in apple juice during storage at 25 °C and 45 °C. Apple juice was inoculated with vegetative cells or spores of A. acidoterrestris, and supplemented with 0, 10, 50, or 100 ppm NG. Half of the samples were subjected to heat treatment at 185°F (85 °C), and the other half were not. Samples were stored at 25 °C or 45 °C for 28 days with samplings on days 0, 1, 3, 7, 14, 21, and 28. The minimum inhibitory concentration of NG was 4 and 8 ppm against spores and vegetative cells, respectively. The minimum bactericidal concentration of NG varied between strains, but was much higher than 100 ppm, which is the maximum recommended concentration for NG in juice and beverage products. During the 28-day storage, all tested concentrations of NG completely inhibited growth and outgrowth of A. acidoterrestris vegetative cells and spores at 45 °C, while the effect of NG was masked at 25 °C. Results also showed heating had some inhibitory effects against vegetative cells but not spores of A. acidoterrestris. In conclusion, we find NG suitable for inhibiting the growth of A. acidoterrestris in shelf-stable apple juice.