Microbial necromass carbon (MNC) serves a crucial function in the formation and stabilization of soil organic carbon (SOC). Although biochar amendment is recognized as a promising approach for enhancing SOC sequestration, its impact on MNC accumulation across the paddy soil profile remains uncertain. Through a 4-year field experiment, this study examined the effect of biochar amendment on MNC accumulation across three soil layers (0–15, 15–30, and 30–45 cm) in a paddy soil profile by combining vertical soil profiling, microbial community dynamics, and biomarker analysis. The results showed that biochar amendment reduced MNC by 10.5% (0–15 cm), 7.5% (15–30 cm), and 9.6% (30–45 cm), respectively, compared to the unamended control. In the topsoil (0–15 cm), the reduction in MNC under biochar amendment was attributed to decreases in both fungal and bacterial necromass carbon (C), whereas in the subsoil (15–45 cm), it primarily resulted from the decrease in bacterial necromass C. Biochar amendment reduced MNC content by decreasing microbial biomass and increasing nitrogen (N) acquisition enzyme activities, mainly due to a shift in the microbial community toward K-strategists and intensified microbial N limitation. This study provides novel insights into the microbially-mediated SOC dynamics in response to biochar amendment.