Rational modification of carbon nanotubes (CNTs) to isolate their specific physical and chemical properties will inform a mechanistic understanding of observed CNT toxicity in bacterial systems. The present study compares the toxicity of commercially obtained multiwalled carbon nanotubes (MWNTs) before and after physicochemical modification via common purification and functionalization routes, including dry oxidation, acid treatment functionalization, and annealing. Experimental results support a correlation between bacterial cytotoxicity and physicochemical properties that enhance MWNT-cell contact opportunities. For example, we observe higher toxicity when the nanotubes are uncapped, debundled, short, and dispersed in solution. These conclusions demonstrate that physicochemical modifications of MWNTs alter their cytotoxicity in bacterial systems and underline the need for careful documentation of physical and chemical characteristics when reporting the toxicity of carbon-based nanomaterials.