This article focuses on a very peculiar habitat, the thin biofilm that covers the surface of rocks, cobbles, sediment grains, leaf litter, and vegetation on a riverbed. Species composition changes over time and depends on environmental conditions and perturbation of water quality. It provides several ecosystem services, contributing to the biogeochemical fluxes and reducing contamination by absorbing the pollutants. Biofilm into the Toce River (Ossola Valley, Piedmont, Italy) was investigated to assess its capacity to accumulate the metals and macroions from the water column. In this preliminary work, we investigated three sample points, in two different seasons. The community composition of biofilm was determined via morphological analysis (diatoms and non-diatoms algal community). We characterize the biofilm, a community of different organisms, from different perspectives. In the biofilm, Hg was analyzed with an automated mercury analyzer, other metals and macroions with inductively coupled plasma mass spectrometry (ICP-MS) (Al, As, Ba, Ca, Cr, Cu, Fe, K, Mg, Mn, Ni, P, Pb, and Zn), and the carotenoid and chlorophyll composition of the photosynthetic organism with HPLC analysis for the primary producers. The results evidence a seasonal pattern in metals and macroions levels in the biofilm, and a significant difference in the biofilm community and in carotenoid composition, suggesting the utility of using the biofilm as an additional bioindicator to monitor the water quality of the river.