Objectives. To characterize for the first time in vivo a novel bismuth-based nanoparticular contrast agent developed for preclinical applications. Then, to design and test in vivo a multi-contrast protocol for functional cardiac imaging using the new bismuth nanoparticles and a well-established iodine-based contrast agent. Approach. A micro-computed tomography scanner was assembled and equipped with a photon-counting detector. Five mice were administered with the bismuth-based contrast agent and systematically scanned over 5 h to quantify the contrast enhancement in relevant organs of interest. Subsequently, the multi-contrast agent protocol was tested on three mice. Material decomposition was performed on the acquired spectral data to quantify the concentration of bismuth and iodine in multiple structures, e.g. the myocardium and vasculature. Main results. In the vasculature, the bismuth agent provides a peak enhancement of 1100 HU and a half-life of about 260 min. After the injection, it accumulates in the liver, spleen and intestinal wall reaching a CT value of 440 HU about 5 h post injection. Phantom measurements showed that the bismuth provides more contrast enhancement than iodine for a variety of tube voltages. The multi-contrast protocol for cardiac imaging successfully allowed the simultaneous decomposition of the vasculature, the brown adipose tissue and the myocardium. Significance. The new bismuth-based contrast agent was proven to have a long circulation time suitable for preclinical applications and to provide more contrast than iodine agents. The proposed multi-contrast protocol resulted in a new tool for cardiac functional imaging. Furthermore, thanks to the contrast enhancement provided in the intestinal wall, the novel contrast agent may be used to develop further multi contrast agent protocols for abdominal and oncological imaging.