This study aimed to explore the characteristics of bongkrekic acid (BA) produced by Burkholderia gladioli pathovar cocovenenans (BGC) and the effects of ambient temperature and humidity on BA production in the fermented glutinous rice flour (FGRF). In the specific environment with various ambient temperatures (10±1, 15±1, 20±1, 25±1, 30±1, 36±1 ℃) and relative humidity (20%, 40%, 60%), The FGRF was incubated with different bacteria counts of BGC (103, 105, 107 CFU/mL), and BA in the FGRF was measured with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The results revealed that BA could be detectable after only one day of incubation. The BA produced and accumulated by BGC with an initial bacteria count of 103 CFU/mL was more than that in the groups with the initial bacteria counts of 105 and 107 CFU/mL (with a difference of 1-3 μg/kg between groups). Lower temperatures (10±1, 15±1, 20±1 ℃) and humidity (20%) were found to reduce the BA production of BGC in the FGRF. To minimize the risk of foodborne BA poisoning, it is recommended to consume freshly prepared FGRF promptly and avoid storing it for extended periods under hot and humid conditions.