Geobacillus kaustophilus is an important food-borne, spore-forming, thermotolerant bacterium. It has a good potential for biotransformation of steroid hormones, such as progesterone and testosterone. In this study, we report G. kaustophilus membrane lipid modifications in response to temperature shock, salinity, incubation time, and pH. Total lipids significantly increased in response to increasing temperature, incubation time, and salt concentration. However, the bacterium presented a significant decrease in the accumulation of total lipids in response to pH shock. The ratio of branched-chain fatty acids/straight-chain fatty acids decreased significantly under all stress conditions. With an increase in temperature, incubation time, and salt concentration, the ratio of iso-fatty acids/anteiso-fatty acids increased significantly, while this ratio remained unaffected by changes in the pH of the growth medium. Our results suggest a modification occurs in the bacterial membrane structure in response to temperature, salinity, incubation time, and pH shock. The variable abiotic stress resulted in a multiple increase in odd-numbered-carbon and low-melting-point anteiso-branched-chain fatty acids, helping the membrane keep its integrity, fluidity, and function for growth of the bacteria under abiotic stress conditions.