Cancer nanotechnology is emerging as one of the promising strategies combining photothermal therapy (PTT) and photoacoustic imaging (PAI) for the treatment of breast cancer and it has received considerable attention in the recent years because it is minimally invasive, prevents damage to non-targeted regions, permits fast recovery, and involves breast cancer imaging. The present study demonstrates multifunctional biocompatible chitosan-polypyrrole nanocomposites (CS-PPy NCs) as novel agents for photoacoustic imaging-guided photothermal ablation of cancer because of their biocompatibility, conductivity, stability, and strong near-infrared (NIR) absorbance. The CS-PPy NCs are spherical in shape and range 26-94 nm in size with a mean value of 50.54 ± 2.56 nm. The in vitro results demonstrated good biocompatibility of CS-PPy NCs, which can be used in PTT for cancer cells under 808-nm NIR laser irradiation. Tumor-bearing mice fully recovered after treatment with CS-PPy NCs and NIR 808-nm laser irradiation compared to the corresponding control groups. Our research highlights the promising potential of using CS-PPy NCs for photoacoustic imaging-guided photothermal ablation of cancer in preclinical animals, which should be verified in future clinical trials.