The connection between litter chemistry and the pathways controlling soil organic matter (SOM) formation and decay in forest ecosystems remains poorly understood, particularly in tropical soils. We addressed this question by incubating samples of a Ferralsol for 200 days with typical forest litter (leaves, twigs, bark, and roots) obtained from ¹³C-enriched Eucalyptus seedlings. Throughout the incubation, we monitored ¹³C/¹²C–CO₂ evolved from the soil to quantify the microbial respiration of the ¹³C-labeled fresh plant litter and of the native SOM. Afterwards, we used density fractionation to obtain particulate organic matter (POM) with density <1.8 g cm⁻³, and the soil material remaining was wet-sieved to obtain SOM with particle-size >53 μm and mineral-associated SOM (MAOM, with particle-size <53 μm). We used solid-state ¹³C-CPMAS-NMR spectroscopy to assess the molecular composition of plant material and SOM fractions and quantified microbial amino sugars in bulk soil using gas chromatography. Our ¹³C/¹²C–CO₂ results indicate that leaves, twigs, and bark (aboveground litter) were respired at higher rates but led to lower degradation of native SOM as compared to root tissues. On average, aboveground litter promoted net C gains in both POM and MAOM, whereas root litter only led to net C gains in POM. Overall, SOM formation via microbial incorporation of aboveground litter through in vivo pathways appears to be more efficient and causes less degradation of native MAOM than roots. Moreover, a reduction in microbial amino sugars in bulk soils suggests that in vivo pathways also favored the formation of POM, which had more microbial-derived protein than forest litter. Therefore, the connection between litter chemistry and the pathways controlling SOM formation in tropical forest ecosystems must be included in a framework that also considers the mineralization of native SOM and the vertical separation of aboveground and belowground C inputs to soils.