Background and aim Biological soil crusts (BSCs) are critical contributors to the soil organic carbon (SOC) pool in fragile ecosystems, though how they drive carbon sequestration in lithologically complex karst remains unclear. Methods This study investigated moss-dominated biocrusts and their underlying soils derived from dolomite and clastic rocks in the karst region of Southwest China, analyzing the distribution characteristics and internal mechanism of SOC and its fractions across different coverage levels. Results The results showed that: (1) Lithology significantly influenced moss biocrust development, with clastic-derived biocrusts exhibiting greater thickness and biomass than dolomite. (2) The content and stock of SOC and its fractions were higher in dolomite, while clastic rocks were more responsive to increased biocrust coverage, with the increments in total organic carbon (TOC) content and stock (TOCs) at high coverage (80-100%) being 2.00- and 2.11-fold greater than in dolomite. (3) Biocrust development increased contributions of > 2 mm aggregates to TOC and its fractions. The content of Polysaccharide-C was dominant in both lithologies, high-coverage biocrusts significantly increased Phenolic-C and Aromatic-C content. (4) Lithology directly regulated SOC functional groups and fraction contents, while biocrusts directly improved soil particle composition and enhanced TOC and its fractions significantly, driving TOC and its fractions to directly and positively affect TOCs (lambda = 0.851, P < 0.01). Conclusion SOC sequestration is influenced by the synergy of lithology and biocrust, where under lithological constraints, biocrust directly contributes to accumulating carbon fractions and improving soil structure, forming a critical pathway for carbon sink formation in karst ecosystems.