We investigated the species-specific phosphorus (P) nutritional strategies in the microphytoplankton community in the Mahon estuary (Minorca, Western Mediterranean) in 2011, under two contrasting hydrographic scenarios. Estuarine flow, nutrient concentrations, phytoplankton community composition, and enzyme-labeled fluorescence (ELF) were measured in June and October, corresponding to the beginning and the end of summer. Dissolved inorganic nitrogen (DIN) and inorganic phosphate (P i ) exhibited enhanced concentrations in the inner estuary where N:P molar ratios suggested P-limitation in both surveys. P i was low and variable (0.09 ± 0.02 µmol L −1 in June and 0.06 ± 0.02 µmol L −1 in October), whereas organic phosphorus remained a more readily available P source. Even though ambient P i concentrations were slightly higher on June, when the microphytoplankton assemblage was dominated by dinoflagellates, the percentage of ELF labeled cells was notably higher (65 ± 9% of total cells) than in October (12 ± 10%), when the presence of diatoms characterized the microphytoplankton community. Alkaline phosphatase activity (AP) was mainly expressed by dinoflagellate taxa, whereas diatoms only displayed significant AP in the inner estuary during the June survey. A P-addition bioassay in which response of AP to P i enrichment was evaluated showed marked reduction in AP with increasing P i . However, some dinoflagellate species maintained AP even when P i was supplied in excess. We suggest that in the case of some dinoflagellate species AP is not as tightly controlled by ambient P i as previously believed. AP activity in these species could indicate selective use of organic phosphorus, or slow metabolic response to changes in P forms, rather than physiological stress to low P i availability. We emphasize the importance of identifying the species-specific phosphorus requirements and their capability for P assimilation in order to understand the contribution of microphytoplankton to the whole community response to biogeochemical perturbations.