Background: Non-alcoholic fatty liver disease (NAFLD) is increasing worldwide. A main cause is the obesogenic, so-called Western lifestyle. NAFLD follows a long, unperceived course, and ends potentially fatally. Early diagnosis of aggressive subtypes saves lives. So far, non-invasive means of detection are limited. A better understanding of the pathogenic interplay among insulin resistance, immune inflammation, microbiome, and genetic background is important. Metabolomics may give insight into these interlaced processes. Methods: In this study, we measured bile acids (BA) in the plasma of adult NAFLD and alcohol-associated liver disease (ALD) patients and healthy controls with targeted mass spectrometry. We focused on gender-related bile acid production pathology in NAFLD and ALD. Results: Compared to healthy controls, women with NAFLD had significantly higher concentrations of total BA, total primary BA, total cholic (CA), total chenodeoxycholic (CDCA), total glycine-conjugated, and total non-12-a-OH BA. Concerning subtypes, glycocholic (GCA) and glycochenodeoxycholic (GCDCA), BA were elevated in women with NAFLD. In contrast, men with NAFLD had no significantly altered total BA fractions. However, the subtypes GCA, glycodeoxycholic (GDCA), glycolithocholic (GLCA), lithocholic (LCA), taurolithocholic (TLCA), and tauroursodeoxycholic acid (TUDCA) were elevated, while CA was significantly decreased. In NAFLD, except ursodeoxycholic acid (UDC), all total BA correlated significantly positively in both sexes with the ELF score, while in ALD, only males showed significant correlations exceptive for total UDC BA. In NAFLD, total BA, total primary BA, total secondary BA, total free secondary BA, total CA, total CDCA, total taurine conjugated, total glycine conjugated, total 12-a-OH, and total non-12-a-OH were significantly higher in cases of a high enhanced liver fibrosis (ELF) score above 9.8. In ALD, total UDC was additionally elevated. Between NAFLD with and without NASH, we found no significant differences. Conclusion: Our data show gender-specific bile acid profiles in NAFLD and markedly different BA patterns in ALD. Women with NAFLD had more severe cholestasis. Men may better compensate fat storage-driven bile acid dynamics, indicated by higher levels of taurine-conjugated BA, which associate with beneficial metabolic functions.