Microphytobenthos are the major intertidal primary producers in many estuarine systems and assessment of their biomass is essential in ecological studies. Algal biomass can be assessed by the use of proxy measurements, e.g. chlorophyll a (Chl a), using a lengthy and destructive extraction procedure. Fluorescence is a non-destructive and in situ alternative, recently applied to microphytobenthos, using the minimum fluorescence signal (Fo) as a measurement of Chl a. In this study, Fo after 15 min of dark adaptation (Fo15) showed a significant positive correlation with Chl a (r = 0.84, p<0.001, n = 24). The relationship between Fo15 and Chl a extracted from sediment samples of increasing sample depth (0.2–2 mm) became less significant as sediment depth increased. The strongest relationship between Fo15 and Chl a, expressed as concentration (mg m−2) and content (mg kg−1), was found when all data were combined, but nearly comparable levels of association were found for in situ measurements using sediment cores up to 2.0 mm deep. Results suggested that full dark adaptation was not always achieved because a significant negative correlation was observed between Fv/Fm and ambient irradiance for seasonal data. In addition, Fo15 showed a more significant correlation with Chl a than with Fm15, suggesting some residual level of non-photochemical quenching after 15 min in the dark. Despite the incomplete dark adaptation, the highly significant relationships between Fo15 and both Chl a concentration and content suggest that the use of Fo15 is a suitable in situ proxy method of estimating algal biomass.