The possible protective role of endogenous isoprene against oxidative stress caused by singlet oxygen (1O2) was studied in the isoprene-emitting plant Phragmites australis. Leaves emitting isoprene and leaves in which isoprene synthesis was inhibited by fosmidomycin were exposed to increasing concentrations of 1O2 generated by Rose Bengal (RB) sensitizer at different light intensities. In isoprene-emitting leaves, photosynthesis and H2O2 and malonyldialdehyde (MDA) contents were not affected by low to moderate 1O2 concentrations generated at light intensities of 800 and 1240 µmol m−2 s−1, but symptoms of damage and reactive oxygen accumulation started to be observed when high levels of 1O2 were generated by very high light intensity (1810 µmol m−2 s−1). A dramatic decrease in photosynthetic performance and an increase in H2O2 and MDA levels were measured in isoprene-inhibited RB-fed leaves, but photosynthesis was not significantly inhibited in leaves in which the isoprene leaf pool was reconstituted by fumigating exogenous isoprene. The inhibition of photosynthesis in isoprene-inhibited leaves was linearly associated with the light intensity and with the consequently formed 1O2. Hence, physiological levels of endogenous isoprene may supply protection against 1O2. The protection mechanisms may involve a direct reaction of isoprene with 1O2. Moreover, as it is a small lipophilic molecule, it may assist hydrophobic interactions in membranes, resulting in their stabilization. The isoprene-conjugated double bond structure may also quench 1O2 by facilitating energy transfer and heat dissipation. This action is typical of other isoprenoids, but we speculate that isoprene may provide a more dynamic protection mechanism as it is synthesized promptly when high light intensity produces 1O2.