Understanding the biogeochemical processes governing carbon (C) and nitrogen (N) cycling in karst aquifers is critical, yet the specific pathways through which dissolved organic matter (DOM) influences nitrogen cycling under varying degrees of aquifer confinement remain poorly understood. This study addresses this knowledge gap by analyzing geochemical characteristics, DOM composition, and microbial communities in three types of aquifer media, fissure groundwater (KFW), conduit groundwater (KCW), and surface water (KSW), at the Zengpiyan karst site in southern China, using carbon isotopes, FT-ICR MS, and amplicon sequencing. Results indicate that hydraulic residence time is the primary factor driving differences in DOM molecular characteristics and biodegradation, with KFW exhibiting conditions favoring denitrification and KCW/KSW supporting nitrification. DOM in karst aquifers is predominantly composed of terrestrial, low NOSC lignin compounds (-0.3 < NOSCwa < -0.1), which degrade in relation to hydraulic retention. Longer residence times enhance degradation of lignin and nitrogenous molecules by key microbial taxa. Potential Molecular Transformation analysis and Spearman correlation network analysis highlight the specific contributions of ASV9 (Comamonas sp.), ASV79 (Nitrospira sp.), and ASV95 (Comamonadaceae sp.) to CHON compound transformation, linking microbial-driven DOM degradation to nitrogen cycling. These findings underscore the interconnected C-N biogeochemical processes in karst aquifers and offer insights into the roles of specific microbial communities in mediating nitrogen transformations, with implications for groundwater remediation in polluted karst systems.