Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide. Although gut microbiota dysbiosis and aberrant bile acid metabolism are central to MASLD pathogenesis, the critical keystone species and precise molecular mechanisms remain elusive. Here, we performed an integrated analysis of 14 public 16S rRNA datasets (275 controls, 352 patients), combining multi-method differential abundance screening, machine learning, and network analysis to pinpoint MASLD-specific microbial signatures. We further validated the therapeutic potential of candidate secondary bile acids (SBAs) in a HepG2 steatosis model. MASLD patients displayed significantly reduced microbial diversity and a distinct community structure. Bifidobacterium longum emerged as the top discriminative putative keystone species, showing marked depletion and reduced network centrality. The secondary bile acid biosynthesis pathway was downregulated and positively correlated with B. longum abundance. Mechanistically, in vitro assays demonstrated that two SBAs, 23-nor-deoxycholic acid (23-NCA) and sodium taurodeoxycholate (TDCA-Na), alleviated hepatocellular lipid accumulation and inflammation, concomitant with TGR5 upregulation, AMPK activation, and p38 MAPK inhibition. In conclusion, depletion of the putative keystone species B. longum is associated with downregulated predicted genomic potential for secondary bile acid biosynthesis, while candidate SBAs protect hepatocytes in association with the activation of the hepatic TGR5–AMPK signaling axis in vitro. These findings offer a mechanistic basis for therapeutic strategies targeting the microbiota-bile acid axis.