This study aimed at evaluating the potential of an in situ algal bioassay for routine toxicity estimates of potentially contaminated estuarine environments using the microalga Phaeodactylum tricornutum immobilized in alginate beads. The influence of the initial cell density in the beads and of salinity on algal growth was first investigated. The potential of the proposed bioassay was evaluated by comparing laboratory with in situ results. A good growth performance of P. tricornutum was observed at all starting densities of beads. Although the growth rate of P. tricornutum was significantly affected by salinity, acceptability criteria currently adopted in algal assays were met, indicating the suitability of P. tricornutum as a test species for bioassays in estuaries. The large differences observed between the laboratory and in situ responses of P. tricornutum were most probably due to the temperature and light conditions less favorable for algal growth in the field and to the lack of representativeness of water samples compared to the field fluctuating conditions. These results showed the need for in situ assessments, especially in estuarine environments influenced by tides. To a lesser extent, the bioassay itself may also have been responsible for the laboratory and field differences. Further improvements in the bioassay chambers and procedures were also discussed.