The formation of reactive species in the afterglow of a radio-frequency-driven atmospheric-pressure plasma in a fixed helium–oxygen feed gas mixture (He+0.5%O2) with humid air impurity (a few hundred ppm) is investigated by means of an extensive global plasma chemical kinetics model. As an original objective, we explore the effects of humid air impurity on the biologically relevant reactive species in an oxygen-dependent system. After a few milliseconds in the afterglow environment, the densities of atomic oxygen (O) decreases from 1015 to 1013 cm−3 and singlet delta molecular oxygen (O2(1D)) of the order of 1015 cm−3 decreases by a factor of two, while the ozone (O3) density increases from 1014 to 1015 cm−3. Electrons and oxygen ionic species, initially of the order of 1011 cm−3, recombine much faster on the time scale of some microseconds. The formation of atomic hydrogen (H), hydroxyl radical (OH), hydroperoxyl (HO2), hydrogen peroxide (H2O2), nitric oxide (NO) and nitric acid (HNO3) resulting from the humid air impurity as well as the influence on the afterglow chemistry is clarified with particular emphasis on the formation of dominant reactive oxygen species (ROS). The model suggests that the reactive species predominantly formed in the afterglow are major ROS O2(1D) and O3 (of the order of 1015 cm−3) and rather minor hydrogen- and nitrogen-based reactive species OH, H2O2, HNO3 and NO2/NO3, of which densities are comparable to the O-atom density (of the order of 1013 cm−3). Furthermore, the model quantitatively reproduces the experimental results of independent O and O3 density measurements.