The effects of a compound probiotic powder containing Bifidobacterium animalis subsp. lactis BLa80 and Lacticaseibacillus rhamnosus LRa05 on the immune function of mice and its acute oral toxicity were investigated. The compatibility of BLa80 and LRa05 was verified via an antagonism assay, and acute oral toxicity was assessed via high-dose gavage in experimental animals, with clinical observations recorded over 14 days, followed by determination of the median lethal dose (LD50). Different doses (33.5, 67, 201 mg•kg-1 BW) of the compound probiotic powder were orally administered to healthy mice for 30 consecutive days, and indicators such as body weight, humoral and cellular immune functions, and natural killer (NK) cell activity were measured. The results showed no antagonism between BLa80 and LRa05, indicating their compatibility for combined use. In the acute oral toxicity study, no signs of poisoning or mortality were observed in male or female mice and rats. The LD50 exceeded 20.0 g•kg-1 BW for both sexes of mice and 10.0 g•kg-1 BW for rats. Compared with the blank control group (52.48), the hemolytic concentration (HC50) value of the high-dose group was significantly elevated (55.18, P<0.05), whereas no significant differences were detected in the low- and medium-dose groups (52.70, 53.68). The carbon clearance capacity was enhanced in both medium- and high-dose groups (5.72, 5.65, P<0.05), and NK cell activity was significantly increased in the high-dose group (56.04%, P<0.05). In conclusion, the compound probiotic powder was found to enhance humoral immunity (high-dose), macrophage function, and NK cell activity. It was also shown to be highly safe and exhibited no acute toxicity, thus providing new ideas and directions for its application in immune regulation therapies.