Semiconductor SnO nanoparticles (NPs) are being exploited for various applications, including those in the environmental context. However, toxicity studies of SnO NPs are very limited. This study evaluated the toxic effect of two sizes of spherical SnO NPs (2 and 40 nm) and one size of flower-like SnO NPs (800 nm) towards the environmental bacteria E. coli and B. subtilis. SnO NPs were synthesized using a hydrothermal or calcination method and they were well characterized prior to toxicity assessment. To evaluate toxicity, cell viability and membrane damage were determined in cells (1 × 10 CFU mL) exposed to up to 1000 mg L of NPs, using the plate counting method and confocal laser scanning microscopy. Spherical NPs of smaller primary size (E2) had the lowest hydrodynamic size (226 ± 96 nm) and highest negative charge (-30.3 ± 10.1 mV). Smaller spherical NPs also showed greatest effect on viability (IC > 500 mg L) and membrane damage of B. subtilis, whereas E. coli was unaffected. Scanning electron microscopy confirmed the membrane damage of exposed B. subtilis and also exhibited the attachment of E2 NPs to the cell surface, as well as the elongation of cells. It was also apparent that toxicity was caused solely by NPs, as released Sn was not toxic to B. subtilis. Thus, surface charge interaction between negatively charged SnO NPs and positively charged molecules on the membrane of the Gram positive B. subtilis was indicated as the key mechanism related to toxicity of NPs.