This study was conducted to investigate the effect of different levels of Protexin on the bacterial load in the gut of Japanese quails. Forty eight Japanese quails (Coturnix japonica) were equally allocated to four treatments containing three pens (include 3 female and 1 male) in each treatment. Birds received diets supplemented with 0, 0.250, 0.500, and 1.000 gm Protexin/kg as treatments 1 (control), 2, 3 and 4 respectively. At the age of 14 weeks, four quails (two male and two female) were randomly selected from each treatment and slaughtered. The carcasses were immediately opened and the entire intestine was removed aseptically. Bacterial load of Campylobacter, Closterdium perfringens and Lactobacillus were measured using Real Time Polymerase Chain Reaction (PCR). Lactobacillus bacteria concentration as beneficial digestive bacteria was the highest in treatment 2 followed by treatments 4 and 3. Concentration of Closterdium perfringens in quails fed treatment 2 had the highest concentration compared to the control. Concentration of Campylobacter in quail’s colon fed treatment 2 had the highest concentration compared to the control group. The results showed that the supplementation of 500 gm/kg Protexin has a beneficial effect on gastrointestinal flora of laying quails.