The microphytobenthos (MPB), a microbial community of primary producers, play a key role in coastal ecosystem functioning, particularly in intertidal mudflats. These mudflats experience challenging variations of irradiance, forcing the micro-organisms to develop photoprotective mechanisms to survive and thrive in this dynamic environment. Two major adaptations to light are well described in literature: the excess of light energy dissipation through non-photochemical quenching (NPQ), and the vertical migration in the sediment. These mechanisms trigger a lot of scientific interest, but the biological processes and metabolic mechanisms involved in light-driven vertical migration remain largely unknown. To our knowledge, this study investigates for the first time metabolomic responses of a migrational mudflat biofilm exposed for 30 min to a light gradient of photosynthetically active radiation (PAR) from 50 to 1000 µmol photons m−2 s−1. The untargeted metabolomic analysis allowed to identify metabolites involved in two types of responses to light irradiance levels. On the one hand, the production of SFAs and MUFAs, primarily derived from bacteria, indicates a healthy photosynthetic state of MPB under low light (LL) and medium light (ML) conditions. Conversely, when exposed to high light (HL), the MPB experienced light-induced stress, triggering the production of alka(e)nes and fatty alcohols. The physiological and ecological roles of these compounds are poorly described in literature. This study sheds new light on the topic, as it suggests that these compounds may play a crucial and previously unexplored role in light-induced stress acclimatation of migrational MPB biofilms. Since alka(e)nes are produced from FAs decarboxylation, these results thus emphasize for the first time the importance of FAs pathways in microphytobenthic biofilms acclimation to light.