The present study investigates the effects of dietary probiotics on the growth performance, feed utilization, and gastrointestinal microflora of Pangasianodon hypophthalmus over a 60-day experimental period. Three probiotic strains Bacillus subtilis, Lactococcus lactis, and Saccharomyces cerevisiae were administered individually and in combination to evaluate their impact on fish growth, nutrient assimilation, and survival. Fish fed with the mixed probiotic diet (T₄) exhibited the highest weight gain (241.84±0.17 g), specific growth rate (1.53±0.57%), feed conversion efficiency (78.13±0.14%), and protein efficiency ratio (2.22±0.11) compared to other treatment groups and control. Gastrointestinal microflora analysis showed significant colonization of probiotic strains, enhancing gut microbial balance and digestive enzyme activity, which contributed to improved nutrient absorption and growth.