The cytotoxicity of Al 2O 3 nanoparticles (NP) at very low exposure levels (1 μg/mL and less) to a dominant bacterial isolate from freshwater (lake water), Bacillus licheniformis, was examined. Sterile lake water was directly used as a test medium or matrix to simulate the freshwater environment. Exposure to 1 μg/mL Al 2O 3 NP for 2 h caused a 17% decrease in cell viability (as determined by plate count and MTT assay). During the test period, the particles were found to be stable against aggregation in the matrix and exerted a nano-size effect on the exposed test organisms. The decrease in cell viability was proven not to be due to the release of Al 3+ ions from the nanoparticles in the dispersion. The zeta potential and FT-IR analyses suggested that the surface charge based attachment of nanoparticles on to the bacterial cell wall was responsible for flocculation leading to toxicity. The cell wall damage confirmed through SEM and the lipid peroxidation assay also contributed toward toxicity. This study warns of possible ecotoxicity of nanoparticles even at environmentally relevant concentrations. However, detailed studies need to be carried out to establish probable mechanistic aspects of this low concentration toxicity phenomenon. © 2011 American Chemical Society.