Microbial fuel cell (MFC) technology holds enormous potential for inexpensive real-time and onsite testing of water sources. With the intent of defining optimal operational conditions, we investigated the effect of environmental factors (changes in temperature, pH and ionic strength), on the performance of a single chamber miniature MFC sensor. The pH of the influent had the greatest effect on the MFC performance, with a 0.531 ± 0.064 μA cm−2 current variation per unit change of pH. Within the range tested, temperature and ionic strength had only a minor impact (0.010 ± 0.001 μA °C−1 cm−2 and of 0.027 ± 0.003 μA mS−1 cm cm−2 respectively). Under controlled operational conditions, for the first time, we demonstrated the ability of this biosensor to detect one of the most commonly applied pesticides worldwide, atrazine. The sensitivity to atrazine was 1.39 ± 0.26 ppm−1 cm−2, with a detection range of 0.05–0.3 ppm. Guidelines for systematic studies of MFC biosensors for practical applications through a factorial design approach are also provided. Consequently, our work not only enforces the promise of miniature MFC biosensors for organic pollutants detection in waters, but it also provides important directions towards future investigations for infield applications.