Ionic liquids (ILs) are novel organic salts with a wide liquid range and have enormous potential for industrial use as green replacements for harmful volatile organic solvents. Varying the cationic components can alter the properties of ILs to suit a variety of industrial applications. However, to complement designer engineering, it is crucial to proactively characterize the biological impacts of new chemicals, in order to fully define them as environmentally friendly. Before industrial release of ILs into the environment, we performed a comprehensive analysis of the toxicity, mutagenicity and microbial biodegradability of ILs in order to provide guidelines for further green chemical synthesis. A broad toxicity analysis was performed on a suite of ILs using the Microtox Acute Toxicity test which determines the effects of a potential toxin to the standard test microorganism, Vibrio fischeri. Using this information, we focused on six imidazolium and pyridinium ILs for further toxicity and mutagenicity analyses. The results indicate that increasing the length of a substituted alkyl chain from butyl to hexyl to octyl on the IL cation causes an increase in toxicity and microbial growth inhibition. However, none of the ILs tested caused frameshift or missense mutations to Salmonella typhimurium in the Ames Test for Mutagenicity, and can be classified as non-mutagenic. Biodegradability by an activated sludge microbial community of the six imidazolium and pyridinium ILs was assessed using the OECD DOC Die-Away Test, and complemented by 1H-NMR analysis and RP-HPLC-MS analysis. We determined that imidazolium-based ILs are highly resistant to biodegradation, but that pyridinium-based ILs can be fully metabolized. In addition, biodegradability is enhanced with longer alkyl chain length substitution on the pyridinium ring. One IL, 1-octyl-3-methylpyridinium bromide is classified as 'readily biodegradable' by OECD standards. The first experimental examination of an IL biodegradation pathway, microorganisms involved in IL biodegradation and toxicity of biodegradation products to Daphnia magna are presented. This research represents an integrated approach, combining expertise in the fields of microbiology, toxicology and chemical engineering, to address the challenges of green chemistry and pollution prevention. This information will aid in the design of novel green chemicals and provide a guide for future collaborative research.