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Little is known about the fate of carbon nanotubes in aquatic environments. To investigate their interactions with dissolved organics and microorganisms, carbon nanotubes (CNTs) were exposed to natural river water in microcosms for 50 days. CNTs and developed biofilms were recovered and examined using scanning transmission X-ray microscopy (STXM). CNTs underwent extensive aggregation, forming bundles and flocs. CNTs were also integrated into developing biofilm and either became associated with the biology or acted as a scaffold for growth. STXM examination revealed the development of an extensive complex coating consisting of lipids, proteins, polysaccharides and carbonates. Single walled-CNT had a significantly greater affinity, on a per-unit material basis, for protein (2× more) and polysaccharides (10× more) relative to multi walled-CNTs. These bio-molecular coatings constitute a highly-modified surface chemistry influencing a range of functional properties, including the toxicity of these nanomaterials. Reactive oxygen species (ROS) are linked to CNTs toxicity; therefore, we compared ROS production in bacteria by “as-manufactured” with that by biofilm-coated CNTs. Fluorescence confocal laser microscopy using carboxy-H2DCFDA demonstrated a significant reduction in ROS production in bacteria exposed to coated CNTs. CNT-based toxicity will likely be rapidly attenuated when these materials enter aquatic environments. These observations contribute to understanding the fate and potential effects of CNTs in natural systems, and confirm the need to evaluate the impact of complex bio-molecular coatings on environmental effects of nanomaterials.