At present, there is an urgent necessity for the discovery of new chemotherapeutic agents with novel molecular skeleton structures that exhibit wide spectrum antitumor activity. In this work, a cationic pentathiophene (5T) is synthesized and discovered to have both anticancer activity and molecular imaging property. 5T can selectively accumulate in mitochondria to exhibit organellar imaging and efficiently induce cell apoptosis associating with JNK pathway activation. Additionally, complexes are prepared through electrostatic interactions between 5T and sodium chlorambucil (a widely used anticancer drug) with varying molar ratios. The complexes form nanoparticles in water with the size of about 50 nm. The 5T-chlorambucil nanoparticles enhance anticancer activity by 2–9 fold due to the synergistical anticancer activity of 5T and chlorambucil. 5T is therefore a promising multifunctional anticancer agent that incorporates optical monitoring capability and anticancer activity that targets mitochondria.