Although silver nanoparticles are the most widespread product of nanotechnology, the mechanisms underlying AgNP microbial toxicity remain the subject of intense debate. In this study, Bacillus subtilis has been used as model organism to elucidate the molecular interactions between this class of bacteria and different forms of silver such as nanoparticles, nanoparticles functionalized with tetracycline and silver ions. For this purpose, we carried out transmission electron microscopy and MALDI-TOF MS analysis of cells treated with silver nanoparticles (AgNPs, AgNPs functionalized with tetracycline, combination of AgNPs with tetracycline) and silver ions as well as we measured the level of reactive oxygen species. The data demonstrate that B. subtilis exhibits high resistance to silver nanoparticles and this phenomenon is associated with following processes: (I) initiation of endospore formation, (II) reduction of free Ag⁺ released from nanoparticles and (III) modification of the AgNPs surface. However, high silver ions concentration appeared to be very toxic to studied strain of bacterium. MALDI-TOF MS analysis revealed that the spectra of B. subtilis cells treated with silver ions are significantly different from spectra of control cells and cell treated with AgNPs and antibiotic which can suggest that silver ions in the highest degree modify bacterial components.