Aims: To study the accumulation of the bacterial living cells (LC) and dead cells (DC) in a mixed‐species biofilm developed in a 3 l biotrickling filter (BTF) challenged with toluene. Methods and Results: The bacterial LC and DC within the biofilm developed on polypropylene Pall rings in a toluene‐degrading BTF were enumerated as fluoro‐microscopic counts during a 62‐operating day period using nucleic acid staining and the direct epifluorescence filter technique. The biofilm development could be separated into three distinct phases: (i) cell attachment, (ii) biofilm establishment and (iii) biofilm maturation. The LC were always dominant (≥72%) in the biofilm during the establishment phase whereas the average LC fraction decreased to 51% of the total cells in the maturation phase. The concentration of LC and DC was observed to level off after 41 days at 1010 cells per ring. The biofilm thickness and the dry weight increased independently of the cell number during the maturation phase. Conclusions: After the LC reached a maximum concentration in the biofilm, the biofilm proliferation was only characterized by the accumulation of DC and organic matter. Significance and Impact of the Study: The results obtained in the present study are of particular relevance for biofilm mathematical modelling and numerical simulations. They will also be useful to estimate the contribution of the living bacteria within the biofilm in bioprocesses.