This study examined the effects of storage conditions such as time course, temperature, fluorescent light, and darkness on the components and antibacterial activity of formalin guaiacol (FG) used in endodontic treatment. We measured the quantities of formaldehyde and guaiacol in FG and antibacterial activities against Staphylococcus aureus, Porphyromonas gingivalis, and Porphyromonas endodontalis. The components and antibacterial activity of FG in the brown or transparent tightly sealed containers were not affected by temperature or fluorescent light throughout the 4 week test. However, in the loosely sealed containers, formaldehyde and guaiacol in FG sample decreased remarkably within one week, not only in a temperature-and time-dependent manner, but also under fluorescent light at 20°C. Furthermore, the antibacterial activities in the FG sample were significantly attenuated in parallel with the decrease in formaldehyde levels. Fluorescent light caused color changes and crystallization of FG samples in the transparent containers. These results suggest that it is important to replace fresh FG every 5 to 7 days for endodontic treatment and that, in the dental office, it is advisable to store fresh FG in tightly sealed containers every 2 weeks to maintain its efficacy.