Climate changes interacting with human activities are raising the temperature in global oceans. To explore physiological responses of in situ phytoplankton assemblages to increasing temperatures, we conducted a shipboard experiment in tropical regions of the eastern Indian Ocean, Java Sea, and southern South China Sea. Throughout the surveyed areas, phytoplankton biomass (Chla) ranged from 0.09 to 0.86 μg L−1 (median, 0.22 μg L−1) in the surface and from 0.30 to 0.99 μg L−1 (median, 0.50 μg L−1) in maximal chlorophyll layer (DCM), respectively. Picophytoplankton that occupied 27–89% (79%) and 83–92% (88%) of total Chla in the surface and DCM layers, ranged from 0.32 × 104 to 23.10 × 104 cells mL−1 (3.69 × 104 cells mL−1) and from 7.44 × 104 to 25.70 × 104 cells mL−1 (12.60 × 104 cells mL−1), respectively. Synechococcus took up 30–97% (78%) of pico-cells compositions in the surface layer, while, in the DCM layer, Prochlorococcus took up 42–98% (91%). Moreover, the maximal photochemical quantum yield (FV/FM) of photosystem II (PS II) and the rapid light curve (RLC)-derived light utilization efficiency (α) were lower in the surface layer than that in the DCM layer, but the saturation irradiance (EK) was higher. In particular, we found that acutely rising temperature decreased the FV/FM and α in both the surface and the DCM layers but increased the absorption cross-section (σPSII) of PSII photochemistry. Our results clearly indicate that the presently rising temperature adversely affects the photophysiology of natural phytoplankton assemblages in tropical oceans.