River biofilms are a suitable indicator of toxic stress in aquatic ecosystems commonly exposed to various anthropogenic pollutants from industrial, domestic, and agricultural sources. Among these pollutants, heavy metals are of particular concern as they are known to interfere with various physiological processes of river biofilm, directly or indirectly related to photosynthetic performance. Nevertheless, only limited toxicological data are available on the mechanisms and toxicodynamics of heavy metals in biofilms. Pulse Amplitude Modulated (PAM) fluorometry is a rapid, non-disruptive, well-established technique to monitor toxic responses on photosynthetic performance, fluorescence-kinetics, and changes in yield in other non-photochemical processes. In this study, a new micro-PAM-sensor was tested to assess potential acute and chronic effects of heavy metals in river biofilm. Toxicity values across the three parameters considered in this study (photosynthetic yield YII, non-photochemical quenching NPQ, and basal fluorescence F0) were comparable, as determined EC50 were within one order of magnitude (EC50 ∼1−10 mg L−1). However, the stimulation of NPQ was more clearly associated with early acute effects, especially in illuminated samples, while depression of YII and F0 were more prevalent in chronic tests. These results have implications for the development of functional indicators for the biomonitoring of aquatic health, in particular for the use of river biofilm as a bioindicator of water quality. In conclusion, the approach proposed seems promising to characterize and monitor the exposure and impact of heavy metals on river periphyton communities. Furthermore, this study provides a fast, highly sensitive, inexpensive, and accurate laboratory method to test effects of pollutants on complex periphyton communities that can also give insights regarding the probable toxicological mechanisms of heavy metals on photosynthetic performance in the river biofilm.