Nanotechnology has been an emerging field due to the promising properties of engineered nanomaterials, materials with at least one dimension less than 100 nanometers. With increasing application of NPs, the risk of these novel materials to environment requires thorough investigation to prevent negative impacts. NPs have enormous variety due to combinations of chemical compositions, sizes, shapes, structures and surface modifications. Building predictive models that link NP properties to biological outcomes is the key to proactive safer NP design. High-throughput toxicity screening and investigating toxicity mechanisms are the common two strategies building towards predictive models of nanotoxicity. These two strategies work together: high-throughput assays facilitate preliminary screening of potentially toxic materials for further mechanistic studies to discover biomarkers and molecular pathways of interest, which will in turn be validated on multiple materials and organisms with high-throughput screening. My thesis work combines both strategies to develop high-throughput screening assays and mechanistic understanding at different molecular levels of how an environmental bacterium, Shewanella oneidensis MR-1, responds to various NP exposures. In this work, Chapter 1 reviews recent advances in analytical nanotoxicology and identifies four key areas that would further bring the field to its maturity. Chapter 2 represents a comprehensive mechanistic study on bacteria responding to TiO2 NPs with UVA illumination. Chapter 3 uses gene expression to explore molecular response among two organisms at different trophic levels to positively and negatively charged gold NPs. Chapter 4 identifies that purification method can be one neglected source of apparent NP toxicity. A high-throughput bacterial viability assay that is free of NP interference is presented in Chapter 5. Finally, in Chapter 6, DNA damage is revealed as a toxicity mechanism for nanoscale complex metal oxide nanomaterials to bacteria.