Obesity is often associated with intestinal microbiota imbalance and increased Gram-negative bacteria characterized by higher endotoxin levels. Therefore, a study on the joint effects of a high-fat diet and Gram-negative bacteria prechallenge might provide further information on the interactive effects of intestinal microbiota and obesity as well as the effects of a probiotic intervention on these processes. This study focused on the joint effects of a high-fat diet and Escherichia coli on mouse inflammatory cytokines, intestinal microbiota, hepatic pathological changes, and the alleviating capacity of probiotics (Lactobacillus plantarum, Bifidobacterium breve, and Lactobacillus fermentum). Our results showed that E. coli administration and the high-fat diet exacerbated the inflammatory syndrome by increasing the visceral fat content, the inflammatory cell infiltration, and intestinal microbiota disorder in mice. E. coli administration caused a decrease in short-chain fatty acids in mouse feces, and probiotics effectively improved this phenomenon. Compound probiotic intervention reduced LPS and IL-1 beta levels, while increased IL-10 levels in mice improved the degeneration and inflammatory infiltration of mouse liver cells. The intestinal microbiota showed great differences at 3 weeks and 6 weeks post-administration. High fat and E. coli alone or in combination caused intestinal microbiota disorder, with increased harmful bacteria, and the probiotics effectively improved the intestinal flora structure and increased the fecal short-chain fatty acid (SCFA) content. In conclusion, a high-fat diet and Gram-negative bacteria challenge exacerbated the inflammatory syndrome, which can be alleviated by compound probiotic intervention.