Isoprene emissions have been considered as a protective response of plants to heat stress, but there is limited information of how prolonged heat spells affect isoprene emission capacity, particularly under the drought conditions that often accompany hot weather. Under combined long-term stresses, presence of isoprene emission could contribute to the maintenance of the precursor pool for rapid synthesis of essential isoprenoids to repair damaged components of leaf photosynthetic apparatus. We studied changes in leaf isoprene emission rate, photosynthetic characteristics, and antioxidant enzyme activities in two hybrid Populus clones, Nanlin 1388 (relatively high drought tolerance) and Nanlin 895 (relatively high thermotolerance) that were subjected to long-term (30 d) soil water stress (25% versus 90% soil field capacity) combined with a natural heat spell (day-time temperatures of 35–40 °C) that affected both control and water-stressed plants. Unexpectedly, isoprene emissions from both the clones were similar and the overall effects of drought on the emission characteristics were initially minor; however, treatment effects and clonal differences increased with time. In particular, the isoprene emission rate only increased slightly in the Nanlin 895 control plants after 15 d of treatment, whereas it decreased by more than 5-fold in all treatment × clone combinations after 30 d. The reduction in isoprene emission rate was associated with a decrease in the pool size of the isoprene precursor dimethylallyl diphosphate in all cases at 30 d after the start of treatment. Net assimilation rate, stomatal conductance, the openness of PSII centers, and the effective quantum yield all decreased, and non-photochemical quenching and catalase activity increased in both control and water-stressed plants. Contrary to the hypothesis of protection of leaf photosynthetic apparatus by isoprene, the data collectively indicated that prolonged stress affected isoprene emissions more strongly than leaf photosynthetic characteristics. This primarily reflected the depletion of isoprene precursor pools under long-term severe stress.