Global warming has been shown to harmfully affect symbiosis between dinoflagellates and other marine invertebrates. When symbiotic dinoflagellates (the genus Breviolum) were in vitro exposed to acute heat stress of +7°C for a period of 5 days, the results revealed the negative impact on all physiological and other cellular parameters measured. The addition of the drug 5-AZA-2’-deoxycytidine (5-AZA), which inhibits DNA methylation processes, was assessed in parallel and contributed to some alterations in algal cellular stress response. Elevated temperatures resulted in a severe reduction in algal density of up to 9.5-fold, as well as pigment concentrations (e.g., chlorophyll a, peridinin, chlorophyll c2, and β-carotene), indicating the status of the physiological stress and early signs of photo-bleaching. Reactive oxygen species (ROS) were increased in all heated dinoflagellate cells, as reported using a highly sensitive flow cytometry methodology. The antioxidant-reduced glutathione (GSH) level initially dropped at day one but increased under prolonged temperature stress. The cell viability parameters were reduced by 97% after 5 days due to an increase in the proportion of apoptotic and necrotic cells overheating time. Autofluorescence (AF) for Cy5-PE 660-20 was reduced from 1.7-fold at day 1 to up to 50-fold drop at the end of heating time, indicating that the AF changes were highly sensitive to heat stress and that it could be an extremely sensitive tool for assessing the functionality of algal photosynthetic machinery. The presence of drug 5-AZA combined with the temperature stress had an additional impact on Symbiodiniaceae density and cell complexity, including the AF levels. Consequently, the increased ROS levels and changes in AF signals reported during ongoing heat stress in dinoflagellate cells could be used as early stress biomarkers in these microalgae and potentially other algal and photosynthetic species.