In vivo tumor targeting and drug delivery properties of small polymerized polydiacetylene (PDA) micelles (∼10 nm) is investigated in a murine MDA-MB-231 xenograft model of breast cancer. Three micelles with different surface coatings are synthesized and tested for their ability to passively target tumor through the enhanced permeability and retention effect. After injection (24 h), fluorescence diffuse optical tomographic imaging indicates a tumor uptake of nearly 3% of the injected dose for the micelles with a 2 kDa poly(ethylene glycol) (PEG)-coating (PDA-PEG2000). The uptake of PDA micelles in tumors is confirmed by co-localization with [18F]-fluorodeoxyglucose (FDG) positron emission tomography. Although FDG has a higher diffusion rate in tumors, 40 ± 19% of the retained micelles is co-registered with the tumor volume visualized by FDG. Finally, PDA-PEG2000 micelles are loaded with the hydrophobic anticancer drug paclitaxel and used in vivo to inhibit tumor growth. These findings demonstrate the potential of PDA-PEG2000 micelles for both in vivo tumor imaging and drug delivery applications.