Intensified coastal eutrophication can result in an overgrowth of seagrass leaves by epiphytes, which is a major threat to seagrass habitats worldwide, but little is known about how epiphytic biofilms affect the seagrass phyllosphere. The physico-chemical microenvironment of Zostera marina L. leaves with and without epiphytes was mapped with electrochemical, thermocouple and scalar irradiance microsensors as a function of four irradiance conditions (dark, low, saturating and high light) and two water flow velocities (approx. 0.5 and 5 cm s−1), which resemble field conditions. The presence of epiphytes led to the build up of a diffusive boundary layer and a thermal boundary layer which impeded O2 and heat transfer between the leaf surface and the surrounding water, resulting in a maximum increase of 0.8°C relative to leaves with no epiphytes. Epiphytes also reduced the quantity and quality of light reaching the leaf, decreasing plant photosynthesis. In darkness, epiphyte respiration exacerbated hypoxic conditions, which can lead to anoxia and the production of potential phytotoxic nitric oxide in the seagrass phyllosphere. Epiphytic biofilm affects the local phyllosphere physico-chemistry both because of its metabolic activity (i.e. photosynthesis/respiration) and its physical properties (i.e. thickness, roughness, density and back-scattering properties). Leaf tissue warming can lead to thermal stress in seagrasses living close to their thermal stress threshold, and thus potentially aggravate negative effects of global warming.