Alzheimer's disease is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NQO1 may accelerate the aggregation of Alzheimer's amyloid beta peptide Ab1-42. Here we examined in-vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Ab1-42 and modify its rate of aggregation. When monitored using the fluorescence of either non-covalent thioflavin T or HiLyte Fluor 647 dye covalently attached to the Ab1-42 peptide, the aggregation kinetics of Ab1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor which binds near the FAD binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Ab1-42. Imaging flow cytometry confirmed in-vitro co-aggregation of NQO1 isoforms and Ab1-42. Ab1-42 alone forms rod shaped fibril structures while in the presence of NQO1 isoforms, Ab1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry analysis indicates that Ab1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of co-aggregates of Ab1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.