In recent years, the study of antimicrobial peptides (AMPs) has garnered considerable attention due to their potential in combating antibiotic-resistant pathogens. Mass spectrometry-based proteomics provides valuable information on microbial stress responses induced by AMPs. This work aims to unravel the proteomic alterations induced by the amyloidogenic antimicrobial peptide R23I, encompassing both inhibitory and non-inhibitory concentrations. This study investigates the effects of the R23I peptide on the protein abundance of Thermus thermophilus (T. thermophilus) at different concentrations (20, 50, and 100 μg/mL). We found 82 differentially expressed proteins, including 15 upregulated and 67 downregulated proteins. We also compared the protein identification results between the PEAKS and IdentiPy programs. Our proteomic analysis revealed distinct patterns of protein expression, suggesting compensatory mechanisms in response to the R23I peptide. Notably, the alterations predominantly affected membrane and cytoplasmic proteins that play a central role in critical cellular processes such as transcription, translation, and energy conversion. This study sheds light on the complex interactions between antimicrobial peptides and bacterial responses, offering insights into microbial adaptability and potential implications for antimicrobial strategies and the understanding of microbial physiology.