Phosphoesterase complex (Pho), a major active component of barley malt, has been demonstrated to be clinically effective in relieving alcoholic fatty liver disease (AFLD), and several lines of evidence have suggested that microbial dysbiosis, caused by chronic alcohol overconsumption, plays a key role in the progression of AFLD. The current study aimed to investigate the modulatory effect of Pho on gut microflora. The microbiota diversity, determined via detection of the V4 region of 16S rDNA genes, was analyzed in rats fed the Lieber-Decarli diet. Gut permeability was evaluated via mucus layer staining. Dysbiosis-associated chronic inflammation was investigated by observing the expression of the following inflammatory molecules in the liver: tumor necrosis factor α (TNF-α), monocyte chemotactic protein 1 (MCP-1), chemokine (C-X-C motif) ligand 1 (CXCL-1) and interleukin 1 beta (IL-1β). Pyrosequencing revealed that the gut microbiota in Pho-treated rats was different from that of AFLD rats at both the phylum and genus levels. In addition, Pho significantly alleviated dysbiosis-associated disruption of gut permeability and inflammation, increased mucus layer thickness and downregulated TNF-α, MCP-1, CXCL-1 and IL-1β expression. In summary, the current results revealed that the microflora, gut barrier and chronic inflammation in AFLD may be modulated by Pho.