Physical (e.g., temperature and light) and biogeochemical (e.g., cycling) parameters are known to affect bioavailability and toxicity of nutrients and pollutants to microorganisms. A system that would allow exposure of selected microorganisms to in situ conditions could provide relevant and novel evaluations of bioavailability. A simple and low cost 37 mL porous underwater chamber (PUC), in which test microorganisms are exposed to field conditions, is presented. The PUC is a thin, acrylic cylinder with polycarbonate membranes on each side, providing an optimal (75 cm2) exchange with the external solution. Regardless of the membrane pore size (0.4 and 5 µm), the PUC required 10 h to equilibrate with the external solution, close to the theoretical time determined for the diffusion of a model compound, the Suwannee River fulvic acid. For in situ use, the PUCs can be filled with filtered water to minimize the equilibration time. The system was validated in 20 to 60 L artificial freshwater by evaluating (1) Cd bioaccumulation by the microalga Chlamydomonas reinhardtii and (2) iron bioavailability to a cyanobacterial bioreporter in comparison with conventional bottle assays. The use of the PUC resulted in no significant differences in Cd uptake fluxes or iron bioavailability. Field experiments undertaken on Lake Erie demonstrated that the PUCs could be used to evaluate the contribution of particulate iron to iron bioavailability. Because the PUCs might not correctly reflect bioavailability for substances at low concentrations with high biouptake fluxes, a critical discussion with respect on the rate-limiting steps of the device is presented. Several considerations that might facilitate the appropriate use of the PUCs are given.