Zinc oxide nanoparticles (ZnO NPs), due to their antibacterial effects, are commonly used in various branches of the economy and can affect rhizobacteria that promote plant growth. We describe the effect of ZnO NPs on two model bacteria strains, B. thuringiensis and B. megaterium, that play an important role in the environment. The MIC (minimum inhibitory concentration) value determined after 48 h of incubation with ZnO NPs was more than 1.6 mg/mL for both strains tested, while the MBC (minimum bactericidal concentration) was above 1.8 mg/mL. We tested the effect of ZnO NPs at concentrations below the MIC (0.8 mg/mL, 0.4 mg/mL and 0.2 mg/mL (equal to 50%, 25% and 12,5% MIC, respectively) in order to identify the mechanisms activated by Bacillus species in the presence of these nanoparticles. ZnO NPs in sublethal concentrations inhibited planktonic cell growth, stimulated endospore formation and reduced decolorization of Evans blue. The addition of ZnO NPs caused oxidative stress, measured using nitroblue tetrazolium (NBT), and reduced the activity of catalase. It was confirmed that zinc oxide nanoparticles in sublethal concentrations change metabolic processes in Bacillus bacteria that are important for their effects on the environment. B. thuringiensis after treatment with ZnO NPs decreased indole acetic acid (IAA) production and increased biofilm formation, whereas B. megaterium decreased IAA production but, inversely, increased biofilm formation. Comparison of different Bacillus species in a single experiment made it possible to better understand the mechanisms of toxicity of zinc oxide nanoparticles and the individual reactions of closely related bacterial species.