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Every surface that is immersed in seawater becomes rapidly covered with an unavoidable biofilm. Such biofilm formation, also known as fouling, is a complex multistage process and not yet thoroughly investigated. In this study, the succession of diatoms and bacteria was investigated during a one month exposure on an artificial substrate of plexiglass (polymer of methyl methacrylate) mounted above the seafloor at a depth of 5 m. For biofilm analyses, the fouling was investigated using selective agar plates, epifluorescence, light and electronic microscopy, as well as high performance liquid chromatography (HPLC) pigment analysis. During biofilm development, the abundance of all biofilm components increased and reached maximum values after a one month exposure. In the bacterial community, heterotrophic marine bacteria were dominant and reached 1.96 ± 0.79 × 104 colony forming units (CFU) cm–2. Despite the fact that faecal coliforms and intestinal enterococci were detected in the water column, faecal coliforms were not detected in the biofilm and intestinal enterococci appeared after one month of exposure but in the negligible number of 60 ± 10 CFU cm–2. The phototrophic component of the biofilm was dominated by diatoms and reached a concentration of 6.10 × 105 cells cm–2, which was supported by pigment analysis with fucoxanthin as dominant pigment in a concentration up to 110 ng cm–2. The diatom community was dominated by Cylindrotheca closterium and other pennate benthic diatoms. A detailed taxonomic analysis by electronic microscopy revealed 30 different taxa of diatoms. The study confirmed that a plexiglass surface in a marine environment is susceptible to biofouling within 30 days of contact. Furthermore, the colonization process sequence firstly involved bacteria and cyanobacteria, and secondly diatoms, which together formed a primary biofilm in the sea.