Using an in-house developed platform, the performance of an Arthrospira maxima biofilm photosynthetic microbial fuel cell (PMFC) was monitored both optically and electrochemically. Fluorescence (excitation wavelength 633 nm, emission range 640 to 800 nm for detection of fluorescence), power density and current output of the PMFC were recorded in real time. Confocal microscopy performed in situ allowed detailed fluorescence imaging to further improve the understanding of the photosynthetic activity of the biofilm that developed on the anode surface of the PMFC, whilst power and current outputs indicated the performance of the cell. The PMFC was shown to be sensitive to temperature and light perturbations with increased temperatures and light intensities resulting in improved performance. A direct relationship between the fluorescent signature and the amount of current produced was identified. With a decreasing external load and increasing current production, the biofilm attached to the anode electrode showed increased fluorescence inferring improved activity of the photosynthetic material. Furthermore, the imaging proved that viable cells covered the entire surface area of the biofilm and that the fluorescence increased with increasing distance (z axis) from the electrode surface.