We have measured directly by time-resolved spectroscopy the transient spectra and kinetics of the methylene blue (MB) excited singlet and triplet state as a function of pH from a few picoseconds to several microseconds. The data show that the acidic triplet state (MBH2+)-M-3 is the protonated analogue of the basic (MB+)-M-3. It is also shown that the singlet oxygen formation quantum yield is much higher in basic than in acidic media. The transient spectra and their kinetics suggest that because pH exerts a large influence in singlet oxygen and radical formation, it may also be important in bacteria inactivation. Therefore, we performed experiments, which showed that the rate of Gram-positive and Gram-negative bacteria inactivation at pH 9 is 3-25 times higher than the rate at pH 5.