The grain aphid Sitobion avenae (Fabricius) is one of the most important cereal pests, damaging crops through sap sucking and virus transmission. Sitobion avenae harbors the secondary endosymbiont Regiella insecticola, which is highly prevalent in populations in south-central Chile and other regions of the world. In order to develop ecological alternatives for biological control, we studied the effect of applying the spores of a strain of the bacterium Bacillus subtilis on the survival and fecundity of the most prevalent genotype of S. avenae in central Chile. The strain selected was one that in previous studies had shown the ability to outcompete other bacteria. Using clones of this aphid genotype infected and uninfected with R. insecticola, we found that applying B. subtilis spores through artificial diets and spraying on leaves decreased both adult survival and nymph production. The detection of spores within the aphid body was negatively correlated with nymph production and was lower in the presence of R. insecticola when applied in diets. B. subtilis spores applied on leaves reduced the number of aphids, an effect that was stronger on aphids harboring R. insecticola. A possible interaction between endosymbiotic bacteria and bacterial antagonists within the aphid body is discussed.