Bombyx mori L. (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV) is a serious pathogen causing huge economic losses to sericulture. There is growing evidence that the gut microbiota of silkworms plays a critical role in shaping host responses and interactions with viral infection. However, little is known about the differences in the composition and diversity of intestinal microflora, especially with respect to silkworm strain differences and BmNPV infection-induced changes. Here, we aim to explore the differences between BmNPV-resistant strain A35 and susceptible strain P50 silkworm and the impact of BmNPV infection on intestinal microflora in different strains. The 16S rDNA sequencing analysis revealed that the fecal microbial populations were distinct between A35 and P50 and were significantly changed post BmNPV infection in both strains. Further analysis showed that the BmNPV-resistant strain silkworm possessed higher bacterial diversity than the susceptible strain, and BmNPV infection reduced the diversity of intestinal flora assessed by feces in both silkworm strains. In response to BmNPV infection, the abundance of Muribaculaceae increased in P50 and decreased in A35, while the abundance of Enterobacteriaceae decreased in P50 and increased in A35. These results indicated that BmNPV infection had various effects on the abundance of fecal microflora in different silkworm strains. Our findings not only broadened the understanding of host–pathogen interactions but also provided theoretical help for the breeding of resistant strains and healthy rearing of silkworms based on symbiotic bacteria.