Since thermophilic microorganisms are valuable source of thermostable enzymes, it is essential to recognize the potential toxicity of silver nanoparticles used in diverse industrial sectors. Thermophilic bacteria Geobacillus vulcani 2Cx, Bacillus licheniformis 3CA, Paenibacillus macerans 3CA1, Anoxybacillus ayderensis FMB1, and Bacillus paralicheniformis FMB2-1 were selected, and their MIC and MBC values were assessed by treating with AgNPs at a range of 62.5-1500 μg mL−1. The growth inhibition curves showed that G. vulcani 2Cx, and B. paralicheniformis FMB2-1 strains were more sensitive to AgNPs, demonstrating a reduction in population by 71.1% and 31.7% at 62.5 μg mL−1 and by 82.9% and 72.8% at 250 μg mL−1, respectively. TEM and FT-IR analysis revealed that AgNPs caused structural damage, cytoplasmic leakage, and disruption of cellular integrity. Furthermore, the cell viability showed a significant decrease alongside an increase in superoxide radicals (SOR; O2·−) production. The β-galactosidase biosynthesis decreased to 28.8% levels at 500 μg mL−1 AgNPs for G. vulcani 2Cx, 32.2% at 250 μg mL−1 for A. ayderensis FMB1, 38.8% only at 62.5 μg mL−1 but completely inhibited at 500 μg mL−1 for B. licheniformis 3CA. Moreover, B. paralicheniformis FMB2-1 showed significant decrease to 11.2% at 125 μg mL−1. This study is the first to reveal the toxic effects of AgNPs on thermophilic bacteria.