Nutrient enrichment may alter patterns of heterotrophic microbial activity (HMA) in wetland soils and influence organic matter decomposition dynamics. The response of the heterotrophic microbial community to C substrates (alcohols, amides, amino acids, aromatics, plant residues, and polysaccharides) was measured as CO 2 and CH 4 production in detritus and soil (0–10 cm) collected from P‐enriched and oligotrophic areas of Water Conservation Area 2a (WCA‐2a) of the Everglades. The wetland was characterized by decreasing P levels from peripheral to interior, oligotrophic areas. Denitrification and SO 4 reduction appeared to be the primary metabolic pathways at the P‐enriched site, whereas the contribution of methanogenesis to organic matter decomposition was greater in the oligotrophic interior of the wetland. Methane production averaged 38 and 48% of the total CO 2 + CH 4 production for P‐enriched and oligotrophic detritus, respectively. Basal CO 2 production of detritus was 36% higher at the P‐enriched than the oligotrophic site, but CH 4 production was 43% greater at the oligotrophic site. All C substrates enhanced CO 2 and CH 4 production, indicating that labile organic C may be limiting in this wetland, and the types of C substrates used by the heterotrophic microbial community varied between P‐enriched and oligotrophic sites. Substrate‐induced respiration was 71 and 48% greater at the P‐enriched than the oligotrophic site for detritus and soil, respectively. Nutrient loading, particularly P, promoted the development of a N‐limited system near the periphery of the wetland, while the oligotrophic interior was characterized by P‐limited conditions. Continued nutrient loading into oligotrophic areas of WCA‐2a may enhance HMA and stimulate organic matter decomposition and nutrient regeneration, and further contribute to undesirable changes to the Everglades ecosystem.