A study of hypericin (Hyp) interaction with mitochondria isolated from U-87 MG glioma cells as well as the time-resolved measurement of singlet oxygen (1O2) formation and annihilation after illumination of the Hyp/mitochondria complex is presented in this work. Interaction between Hyp and mitochondria was studied by steady-state and time-resolved UV–vis absorption and fluorescence spectroscopy. A high concentration of Hyp leads to the aggregation of this compound inside the mitochondria and the relative population of the monomeric (biologically active) form of Hyp decreases concomitantly to approximately 10% at the highest used Hyp bulk concentration. Photosensitized production of 1O2 in mitochondria after illumination of the Hyp/mitochondria complex is characterized by a rise lifetime of 8 s and shows saturation behaviour with respect to Hyp concentration. The lifetime of 1O2 depends on the composition of the medium where the mitochondria are suspended, ranging from about 3.0 s in pure water to 26 s in H2O–D2O (1:9) phosphate buffer. Our results confirm that only the monomeric form of Hyp is able to produce its excited triplet state, which consequently leads to 1O2 production. An influence of photoactivated Hyp on the mitochondria respiration chain was evaluated by the monitoring of time-resolved NAD(P)H fluorescence. We have demonstrated the rise of the NAD(P)H content after illumination of the Hyp/mitochondria complex.