Methanol (MOH) and monomethylamine (MMA) are two typical one-carbon (C1) compounds found in natural environments. They play an important role in marine and atmospheric chemistry, cloud formation, and global climate. The main biological sink of MOH and MMA is rapid consumption by marine microbes. Here, field-based time-series incubations with supplemental 13C-labelled MOH and MMA and isotope ratio analyses were performed. A substantial difference in the MOH and MMA incorporation rates and bacterial taxa were observed between the South China Sea (SCS) and the Pearl River estuary. C1 substrates were assimilated more quickly in the estuary than the SCS shelf where MOH and MMA had similar bio-availability. However, microbial responses to MMA may be faster than to MOH in the coastal and basin surface water of the SCS despite similar active bacterial populations. Three ecological types of bacteria, in terms of response to supplemented MOH and MMA, were identified: rapid incorporation (I, dominant C1-incorporating group), slow incorporation (II, minor C1-incorporating group), and no incorporation (III, C1-non-incorporating group). Members of the families Methylophilaceae (β-Proteobacteria) and Piscirickettsiaceae (γ-Proteobacteria) belonged to type I and actively incorporated substrates in the estuary and SCS, respectively. Diverse MOH and MMA-incorporating type II bacteria were identified by stable isotope probing in the SCS, and could play a more important role in the transformation of C1 compounds in marine environments than hitherto assumed.