This research explores the impact of acute total body irradiation on the gut microbiota of laboratory rats. Ionizing radiation at doses equal to or exceeding 1 Gy is known to induce severe pathological effects, with the hematopoietic system, gastrointestinal tract, and central nervous system being the most vulnerable, depending on exposure level. The primary objective of the study was to evaluate the viability and persistence of key commensal bacteria—Bifidobacterium spp., Lactobacillus spp., and Enterococcus spp.—in the large intestine following acute irradiation. The results revealed a marked, progressive decline in the populations of these beneficial microbes in the irradiated animals, with microbial counts decreasing by a factor of 2.3 to 5.3 by the ninth day compared to day five post-irradiation. Conversely, in the experimental group pre-treated with the probiotic formulation “Lactopropolis-AWL,” the depletion of gut microbiota was significantly attenuated, and bacterial counts remained comparatively stable over time. Moreover, survival rates in this group were substantially higher (88.89%) than those observed in the untreated irradiated group (66.69%). These findings underscore the harmful effects of acute ionizing radiation on the intestinal microbiome and demonstrate the potential of pre-irradiation probiotic intervention as a protective strategy to preserve microbiota integrity and enhance survival.