AbstractGut microbiota has been demonstrated to be involved in intestinal nutrition, defense, and immunity, as well as participating in disease progression. This study was to investigate gut microbiota changes in chickens challenged with netB-positive Clostridium perfringens strain 1 (CP1) and/or the predisposing Eimeria species (Eimeria). In addition, the effects of lauric acid, a medium-chain fatty acid (MCFA), on NE reduction and modulation of microbiota were evaluated. The results demonstrated that microbial communities in the jejunum were distinct from those in the cecum, and the microbial community change was more significant in jejunum. Challenge of CP1 in conjunction with Eimeria significantly reduced species diversity in jejunal microbiota, but cecal microbiota remained stable. In the jejunum, CP1 challenge increased the abundance of the genera of Clostridium sensu stricto 1, Escherichia Shigella, and Weissella, but significantly decreased the population of Lactobacillus. Eimeria infection on its own was unable to promote NE, demonstrating decrements of Clostridium sensu stricto 1 and Lactobacillus. Co-infection with CP1 and Eimeria reproduced the majority of NE lesions with significant increment of Clostridium sensu stricto 1 and reduction in Lactobacillus. The changes of these two taxa increased the severity of NE lesions. Further analyses of metagenomeSeq, STAMP, and LEfSe showed significant overgrowth of Clostridium sensu stricto 1 was associated with NE and Eimeria infection than C. perfringens challenge alone. The supplementation of lauric acid did not reduce NE incidence and severity but decreased the relative abundance of Escherichia Shigella. In conclusion, significant overgrowth of Clostridium sensu stricto 1 in the jejunm is the major microbiota contributory to NE. Controlling proliferation of this taxon in the jejunum should be the niche for developing effective strategies against NE.