Initial biofilm formation was studied during start‐up of anaerobic fixed‐bed reactors with polypropylene as substratum. Five reactors were operated in parallel at 35°C with synthetic wastewater containing acetate, propionate and butyrate as substrates varying the mode of system operation. System variations included (a) operation with recirculation, (b) continuous and discontinuous (fed‐batch) feeding, and (c) different organic loading rates (OLR). An enrichment culture from sludge of a municipal sewage treatment plant, grown in a stirred tank reactor at an OLR of 2g.l−1d−1 for 72 d, was used as inoculum. Reactor performance and biofilm formation were followed over a period of 58 d. Highest biomass accumulation and maximal OLR of 23 kg CODm−3d−1 were obtained in the reactor with the highest space loading. The polypropylene support acted not only as a substratum for biofilm growth but also as a filter retaining biomass in flocs. Scanning electron microscopy (SEM), phase contrast microscopy and epifluorescence microscopy documented various bacteria in the biofilm community and the floc population. Propionic acid and butyric acid oxidation were carried out by Desulfobulbus sp., and by Syntrophomonas sp. and Syntrophospora sp. respectively. Methanothrix sp. was the main acetotrophic methanogen in both floes and biofilm. Morphologically distinct Methanosarcina sp. were only retained in a network of Methanothrix filaments within flocs. Irregular coccoid methanogens of the genera Methanogenium or Methanoculleus were the main hydrogenotrophic methanogens with cells of Methanospirillum sp., Methanocorpusculum sp. and Methanobrevibacter sp. occurring in lower numbers. Bacteria adhering during early phases of biofilm formation were detected as settlers responsible for primary colonisation and initiating development of a mature biofilm. Formation of microcolonies of trophic sub‐populations is demonstrated. Evidence for Desulfovibrio sp. responsible for glycocalyx secretion was provided.