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