The formation of mineral-protected organic carbon (MPOC) is a vital process for soil organic carbon (SOC) accumulation and stabilization, influenced by factors such as exogenous carbon (C) input and soil microorganisms. However, the dynamics of MPOC and soil microorganisms following exogenous C input, and the key microorganisms driving MPOC formation, remain poorly understood. To address this, we conducted exogenous C addition culture experiments to investigate changes in MPOC and soil microorganisms and identify the primary microorganisms influencing MPOC formation. We observed that the MPOC content in treated soils increased over time, ranging from 0.43 to 2.06 g kg−1. MPOC showed a significant positive correlation with soil bacterial diversity and a significant negative correlation with fungal diversity. Soil samples contained 248 bacterial families and 189 fungal genera, with Oxalobacteraceae (7.42%) and unclassified_k__Fungi (24.82%) being the most abundant, respectively. Using FAPROTAX and FunGuild ecological function prediction methods, we analyzed soil bacteria and fungi functional profiles and abundances. We identified the main bacterial families influencing MPOC formation as Microbacteriaceae, Mycobacteriaceae, Pseudomonadaceae, Streptomycetaceae, and Xanthomonadaceae. The primary fungal genera were Cylindrocarpon, Leohumicola, Metarhizium, Neobulgaria, Neopestalotiopsis, Olpidium, and Tetracladium. These findings provide theoretical support for understanding microbial regulation mechanisms in soil C sequestration and emission reduction.