Diatoms are among the main primary producers in marine benthic ecosystems, where they colonize a variety of substrates. Their complex interactions with co-occurring microorganisms and macroorganisms are possibly mediated by infochemicals, including volatile organic compounds (VOCs).  However, the overall VOC emission profile (volatilome) of epiphytic diatoms remains largely understudied. We performed a comprehensive volatilome profiling of two strains of Mastogloia fimbriata, originating from a naturally acidified site and a non-acidified site, respectively, under low irradiance (5 µmol photons m⁻² s⁻¹) and high irradiance (100 µmol photons m⁻² s⁻¹) conditions. Our aim was to understand whether diatoms undergoing long-term adaptation to ocean acidification also exhibit changes in their volatilome. VOCs were analyzed using Proton Transfer Reaction–Time-of-Flight Mass Spectrometry (PTR-ToF-MS), a highly sensitive, real-time analytical technique. The resulting mass spectral fingerprints were evaluated using Principal Component Analysis (PCA), heatmaps and analysis of variance (ANOVA) with post-hoc tests. Distinct volatilome signatures were observed for each strain, indicating that strains selection in acidified sites is associated with significant metabolic reprogramming. Light availability also influenced VOC emissions. This investigation provides the first comprehensive analysis of the volatilome of M. fimbriata and forms a basis for using VOCs as non-invasive diagnostic biomarkers for monitoring benthic ecosystem health and cellular responses to global environmental changes.