Evidence is accumulating to suggest that 3,4-methylenedioxymethamphetamine (MDMA) has neurotoxic and neuroinflammatory properties. MDMA is composed of two enantiomers with different biological activities. In this study, we evaluated the in vivo effects of S(+)-MDMA, R(−)-MDMA, and S(+)-MDMA in combination with R(−)-MDMA on microglial and astroglial activation compared with racemic MDMA, by assessment of complement type 3 receptor (CD11b) and glial fibrillary acidic protein (GFAP) immunoreactivity in the mouse striatum, nucleus accumbens, motor cortex, and substantia nigra. Motor activity and body temperature were also measured, to elucidate the physiological modifications paired with the observed glial changes. Similar to racemic MDMA (4 × 20 mg/kg), S(+)-MDMA (4 × 10 mg/kg) increased both CD11b and GFAP in the striatum, although to a lower degree, whereas R(−)-MDMA (4 × 10 mg/kg) did not induce any significant glial activation. Combined administration of S(+) plus R(−)-MDMA did not induce any further activation compared with S(+)-MDMA. In all other areas, only racemic MDMA was able to slightly activate the microglia, but not the astroglia, whereas enantiomers had no effect, either alone or in combination. Racemic MDMA and S(+)-MDMA similarly increased motor activity and raised body temperature, whereas R(−)-MDMA affected neither body temperature nor motor activity. Interestingly, the increase in body temperature was correlated with glial activation. The results show that no synergism, but only additivity of effects, is caused by the combined administration of S(+)- and R(−)-MDMA, and underline the importance of investigating the biochemical and behavioral properties of the two MDMA enantiomers to understand their relative contribution to the neuroinflammatory and neurotoxic effects of MDMA.