Insect gut microbes played important roles in host feeding, digestion, immunity, growth, and development. The use of antibiotics to remove or inhibit gut bacteria is a commonly used method to study the bacterial gut function of insects. In the present study, the effects of different concentrations of antibiotic mixture on the biological aspects, diversity of the midgut bacteria, and immunity of S. litoralis larvae were examined. The larvae were reared on three different diets. The results indicated that administration of the antibiotic mixture at concentrations ranging from 62 to 500 μg/mL significantly increased the survival rate within the three diets. While the pupal eight was significantly decreased in all diets. The number of midgut bacteria was significantly reduced with the increase of antibiotic concentrations. The gut bacterial diversity of S. litoralis larvae was surveyed and comprised of Bacilli and Gammaproteobacteria on larvae reared on the three diets. While Betaproteobacteria restricted only within the midgut of larvae reared on castor bean leaves. Also, when larvae reared on a diet amended with 500 µg/mL of antibiotic mixture, the midgut bacteria disappeared, and a reduction of cellular immunity occurred. Finally, the results confirm that the use of antibiotics could develop control strategies to get rid of harmful insects without damaging of environment or beneficial organisms by removing the midgut bacteria of pest that are responsible for increasing the immune system.