High ambient temperature is a major problem in commercial broiler production in the humid tropics because high producing broiler birds consume more feed, have higher metabolic activity, and thus higher body heat production. To evaluate the effects of two previously isolated potential probiotic strains (Lactobacillus pentosus ITA23 and Lactobacillus acidophilus ITA44) on broilers growing under heat stress condition, a total of 192 chicks were randomly allocated into four treatment groups of 48 chickens each as follows: CL, birds fed with basal diet raised in 24 °C; PL, birds fed with basal diet plus 0.1 % probiotic mixture raised in 24 °C; CH, birds fed with basal diet raised in 35 °C; and PH, birds fed with basal diet plus 0.1 % probiotic mixture raised in 35 °C. The effects of probiotic mixture on the performance, expression of nutrient absorption genes of the small intestine, volatile fatty acids (VFA) and microbial population of cecal contents, antioxidant capacity of liver, and fatty acid composition of breast muscle were investigated. Results showed that probiotic positively affected the final body weight under both temperature conditions (PL and PH groups) compared to their respective control groups (CL and CH). Probiotic supplementation numerically improved the average daily gain (ADG) under lower temperature, but significantly improved ADG under the higher temperature (P < 0.05) by sustaining high feed intake. Under the lower temperature environment, supplementation of the two Lactobacillus strains significantly increased the expression of the four sugar transporter genes tested (GLUT2, GLUT5, SGLT1, and SGLT4) indicating probiotic enhances the absorption of this nutrient. Similar but less pronounced effect was also observed under higher temperature (35 °C) condition. In addition, the probiotic mixture improved bacterial population of the cecal contents, by increasing beneficial bacteria and decreasing Escherichia coli population, which could be because of higher production of VFA in the cecum, especially at heat stress condition. The two Lactobacillus strains also improved the fatty acid profile of meat, including at heat stress. Generally, the two Lactobacillus strains can be considered as good potential probiotics for chickens due to their good probiotic properties and remarkable efficacy on broiler chickens.