Fluids from the diffusive hydrothermal vent Woody located at the Menez Gwen hydrothermal field were collected directly from a fissure using a Kiel pumping system (KIPS). The fluids were incubated in a biodegradation experiment with the resident microbial community at in situ temperature (55 °C). In addition to the initial fluid (T0), sub-samples were collected at three time points: 12.5 h (T1), 38 h (T2) and 180 h (Tfinal). Whole water samples were characterized by spectrophotometry and for dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) concentrations. Solid-phase extracted dissolved organic matter (SPE-DOM) was molecularly characterized using a 15 Tesla Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR-MS). Based on Mg2 + concentrations, vent fluids were diluted ca. 1:99% with seawater. SPE-DOC concentrations remained constantly low (30 μM) over the course of incubation, while DOC and TDN (1180 μM and 16 μM, respectively, at T0) decreased sharply (to 80 μM and 9 μM, respectively, at Tfinal). This biolabile DOC is likely composed of volatiles and short-chain hydrocarbons, and the reason behind their extraordinarily high concentration remains unknown. Molecular characterization of SPE-DOM in fluids and seawater identified over 3000 molecular formulae, which were separated into different groups according to their reactivity. The half-life of the more reactive groups ranged between 6–7 h, 18–21 h and 5–106 days, and represented 7.3, 2.0 and 7.5% of the total formulae present in T0, while the majority (83%) did not exhibit any significant trend during the experiment. Despite the small molecular changes in SPE-DOM, a decrease in the number of formulae and average molecular mass was observed, the latter consistent with an increase in the spectral slope over the wavelength range 275–295 nm (S275–295) during the experiment. An increase in aromatic molecular formulae during the incubation was also corroborated by a concurrent increase in the absorption coefficient at 254 nm normalized to DOC concentrations (SUVA254), an optical indicator of aromatic content. Thus both FT-ICR-MS and optical properties over the course of the incubation corroborated a decrease in molecular mass and increase in aromaticity. This study is the first to report the optical and molecular transformations of bio-labile DOM from diffusive hydrothermal systems.