Litter burning and biological decomposition are oxidative processes co-occurring in many terrestrial ecosystems, producing organic matter with different chemical properties and differently affecting plant growth and soil microbial activity. We tested the chemical convergence hypothesis, i.e., materials with different initial chemistry converge toward a common profile, with similar biological effects, as the oxidative process advances, for burning and decomposition. We compared the molecular composition, assessed by 13C NMR, of seven plant litter types either fresh, decomposed for 30, 90, 180 d in a microcosms incubation experiment, or heated at 100°C, 200°C, 300°C, 400°C, 500°C for 30 minutes. We used litter water extracts (5% dry weight) as treatments in bioassays on plant (Lepidium sativum) and fungal (Aspergillus niger) growth, and a washed quartz sand amended with litter (0.5% dw) to assess heterotrophic respiration by flux chamber (i.e., [μg of CO2 released]·[g added litter]−1·d−1). We observed different molecular variations for materials either burning (i.e., a sharp increase of aromatic C and a decrease of other fractions above 200°C) or decomposing (i.e., early increase of alkyl, methoxyl, and N-alkyl C and decrease of O-alkyl and di-O-alkyl C fractions). Soil respiration and fungal growth decreased with litter age and heating severity, down to 20% relative to fresh litter. Plants were inhibited on fresh litter (on average 13% of the control), but recovered on aged (180 d) and heated (30 min at 500°C) materials, up to 126% and 63% of the control, respectively. Correlation between the intensity of 13C NMR signals in litter spectra and bioassay results showed that O-alkyl, methoxyl, and aromatic C fractions are crucial to understand organic matter effects, with plant response negatively affected by labile C but positively associated to lignification and pyrogenic C. The pattern of association of soil respiration and fungal growth to these C fractions was essentially opposite to that observed for plant root growth. Our findings suggest a functional convergence of decomposed and burned organic substrates, emerging from the balance between the bioavailability of labile C sources and the presence of recalcitrant and pyrogenic compounds, oppositely affecting different trophic levels.