This research was aimed at investigating the bioactivity of lysozyme towards Alicyclobacillus acidoterrestris. Lysozyme (0.1–20 ppm) was tested towards five different strains; the experiments were performed on both spores and vegetative cells using a microdilution approach to assess the minimal inhibitory concentration (MIC). Thereafter, spore viability of two selected strains was evaluated through the traditional plate count. Finally, an experiment was run to assess the role of lysozyme on the complex phenomenon of spore germination. The MIC of lysozyme towards vegetative cells varied from 0.1 to 6 ppm, while for the spores, it was from 0.1 to 3 ppm. Concerning spore viability, the effect of lysozyme relied upon its amount on the broth, as at MIC it caused a slight reduction in spores (approximately 1 log cfu ml−1) after 24 h; otherwise, higher concentrations caused a decrease in spore level below the detection limit. Concerning spore germination, lysozyme exerted a promoting effect on this phenomenon and reduced the optical density by 66%.