The mitochondrion is one of the key organelles for maintaining cellular homeostasis. External environmental stimuli and internal regulatory processes may alter the metabolism and functions of mitochondria. To understand these activities of mitochondria, it is critical to probe the key metabolic molecules inside these organelles. In this study, we used label-free chemical imaging modalities including coherent anti-Stokes Raman scattering and multiphoton-excited fluorescence to investigate the mitochondrial activities in living cancer cells. We found that hypothermia exposure tends to induce fatty-acid (FA) accumulation in some mitochondria of MIAPaCa-2 cells. Autofluorescence images show that the FA-accumulated mitochondria also have abnormal metabolism of nicotinamide adenine dinucleotide hydrogen, likely induced by the dysfunction of the electron transport chain. We also found that when the cells were re-warmed to physiological temperature after a period of hypothermia, the FA-accumulated mitochondria changed their structural features. To the best of our knowledge, this is the first time that FA accumulation in mitochondria was observed in live cells. Our research also demonstrates that multimodal label-free chemical imaging is an attractive tool to discover abnormal functions of mitochondria at the single-organelle level and can be used to quantify the dynamic changes of these organelles under perturbative conditions.