The availability of rapid and effective methodologies for assessing lotic systems with microphytobenthos is still quite scarce. Hence, the primary goal of this study was to optimize the growth conditions of the sensitive and ubiquous benthic diatom Navicula libonensis for laboratorial and field assessments. The effect of different conditions of temperature, photoperiod, initial cell density, test duration and cell encapsulation into calcium alginate beads was evaluated in a first set of experiments. There was a slight increase in the growth of free and immobilized cells at 23 °C, at lower initial cell densities and at the shortest experimental period (6 days). Through all the conditions, the growth profiles of free versus immobilized were fairly variable. A second experimental trial involved the validation of selected conditions, applied to the ecotoxicological testing of N. libonensis to two reference chemicals-3,5-dichlorophenol and potassium dichromate. A similar response of free and immobilized cells was observed between exposures to spiked stream water and synthetic medium, and through the conditions tested. This outcome suggests that N. libonensis may potentially provide reliable responses under direct in situ exposures.