The present experiment was carried out to investigate the effect of effective microorganisms, EM; carbohydrate sources as molasses, M; wheat bran, and their mixture in a Bio-floc (BF) system on microbial protein production, water quality, growth performance, and feed utilization of Nile tilapia (Oreochromis niloticus) fingerlings. Seven experimental treatments were designed as following: T1: control, fish were fed a basal diet without any addition of carbohydrate source. T2: T1 plus effective microorganisms (EM) in water media. T3: T1 with supplementary molasses (M) in water media. T4: T1 with supplementary wheat bran (WB) in water media. T5: T1 with supplementary of mixture from EM and M in the water media. T6: T1 with supplementary of mixture from EM and WB in the water media, and T7: T1 with supplementary of mixture from M and WB in the water media. Fish in each tank were fed with one of the experimental diets containing 22.49 % crude protein and 471.60 Kcal. gross energy /100g DM at feeding rate of 3% of the actual fish live body weight, two times daily (9.00am and 2.00 pm) for six days a week for 90 days experimental period. Fish were distributed randomly into fourteen indoor fiberglass tanks. Each treatment was conducted in two indoor circular fiberglass tanks of 1m diameter filled with 1000 litter of fresh water. Nile tilapia fingerlings with an average initial body weight of 47.5±0.05 (g/fish) were randomized stocked at the rate of 42 fish / tank. The results showed the followings:The water quality parameters showed no significant differences in water temperature (C⁰),  pH value and DO (mg/l); however, there were marked differences in the values of total ammonia nitrogen (TA-N, mg/l) and Bio-floc (BF) concentrations (mg/l), with high values of TA-N and BF in tanks' water supplemented with different carbohydrate sources than control, especially with T6 which containing mixture of EM and WB in water media followed by T4 (WB),  T3 (M),  T5 (EM+M), T2(EM), and T6 (M+WB), respectively compared  with T1 (control without carbohydrate source).Fish growth performance and feed utilization were significantly (P≤ 0.05) increased with EM and carbohydrate source supplementation than control. Also, T6 which containing a mixture of EM and WB gave the highest performance followed by WB (T4) and M (T3), respectively.Finally, the present results recommended the use of a mixture of effective microorganisms and wheat bran as a carbohydrate source in fish water tanks for optimal performance with best impacts on intensive tilapia culture reared under zero water exchange system.