Diatoms as important phytoplankton components interact with and are colonized by heterotrophic bacteria. Even though this colonization has been studied extensively in the past little if any distinction was made between the bacterial colonization of diatom cells and the aggregated organic material, exopolymeric substances (EPS). Here we show that the diatom Thalassiosira rotula and EPS were differently colonized by two strains of Roseobacteraceae (Planktotalea frisia, Pseudophaeobacter sp. TrK17), one strain of Flavobacteriaceae (Gramella forsetii) in two and three partner treatments and by a natural bacterial community over 21 days. In the two partner treatments with one strain and the diatom the alga and EPS were generally less colonized than in the three partner treatments with two bacterial strains and the diatom. The majority of the bacterial cells, however, remained free-living. Pseudophaeobacter sp. and G. forsetii benefitted greatly by the presence of another partner as the proportions of their subpopulations colonizing the diatom cell and the EPS were much enhanced relative to their two partner treatments. Highest proportions of bacteria colonizing the diatom and EPS occurred in the treatment with the natural bacterial community as the majority of Flavobacteriales and 40% of Roseobacteraceae were associated to the diatom and EPS. Dissolved organic carbon, amino acids and carbohydrates produced by T. rotula were differently used by the bacteria in the two and three partner treatments and most efficiently by the natural bacterial community. Our approach is a valid model system to study physico-chemical bacteria-diatom interactions with increasing complexity.