A natural microphytobenthic assemblage from the Eden Estuary, Scotland, was used to study the effect of temperature and irradiance on the sustainability and species composition of a natural transient biofilm. Three tidal tanks were maintained: tank 1 at 10 °C; tank 2 at 18 °C; and tank 3 at 26 °C. Within each tank, five cores were unshaded (350 μmol/m2 per s), five cores were semi-shaded (175 μmol/m2 per s), and five cores were shaded (70 μmol/m2 per s). Chlorophyll a increased in all treatments, but accumulated slower with increasing temperature. Surface diatom biomass of biofilms (as determined from measurements of minimum fluorescence, Fo15), grown at 10 °C and 18 °C was sustained above initial levels after 21 days, whilst biofilms grown at 26 °C suffered a severe loss of diatoms after 14 days, probably owing to nutrient limitation. Species richness and diversity of diatom assemblages illustrated a variety of responses to the temperature and light conditions. Diatoms at 10 °C acclimated to the different light levels by varying the ratios of diadinoxanthin: chlorophyll a, whilst at 18 °C the diatom species composition changed dramatically in response to shading. Ratios of zeaxanthin: chlorophyll a increased with increasing temperature, indicating an increase in cyanobacterial biomass at 26 °C after 21 days. Increased temperature significantly increased the maximum theoretical electron transport rate (rETRmax) in the short term (days), although light and temperature treatments did not affect the maximum light utilization coefficient (αrETR). Limitations of the fluorescence methodology used to study the resultant mixed community of benthic phototrophs is discussed. Diatom vertical migration in response to light, and an alteration of the main functional taxonomic group, has implications for the interpretation and value of fluorescence data.