The temporal emission characteristics of nile red (NR) single molecules in and on hydrophilic and hydrophobic polymer films are studied as a function of ambient relative humidity (RH), and hence, the film hydration level. The materials studied include poly(vinyl alcohol) (PVA) and poly(methyl methacrylate) (PMMA). The temporal emission characteristics for NR in and on PVA are shown to be. very sensitive to the ambient RH. Autocorrelation of the single-molecule fluorescence transients reveal signal fluctuations on two distinct time scales: 0.1-1 s and 1-2 ms. The fast fluctuations become slower and the slow fluctuations faster with increasing RH. In stark contrast, data obtained from NR in and on PMMA show these films to be almost completely insensitive to ambient moisture. These results point to the important role played by material hydrophilicity in the sensitivity of single-molecule experiments to RH variations. The chemical and physical origins of the RH-dependent signal fluctuations are explored in detail.. Triplet blinking is shown to be the dominant contributor to the fast signal fluctuations for dry films only. Rotational and translational molecular motions are observed to occur more frequently at higher RH. However, it is shown they are not the dominant causes of most signal fluctuations. It is concluded most of the signal fluctuations involve RH-dependent variations in the local molecular environment, which lead to time-dependent variations in the NR emission yield. These variations result from participation of a nonfluorescent twisted intramolecular charge-transfer state in the most polar, hydrated film regions.