Interactions between phytoplankton and bacteria play a central role in mediating biogeochemical cycling and food web structure in the ocean. The cosmopolitan diatoms Thalassiosira and Chaetoceros often dominate phytoplankton communities in marine systems. Past studies of diatom-bacterial associations have employed community-level methods and culture-based or natural diatom populations. Although bacterial assemblages attached to individual diatoms represents tight associations little is known on their makeup or interactions. Here, we examined the epibiotic bacteria of 436 Thalassiosira and 329 Chaetoceros single cells isolated from natural samples and collection cultures, regarded here as short-and long-term associations, respectively. Epibiotic microbiota of single diatom hosts was analyzed by cultivation and by cloning-sequencing of 16S rRNA genes obtained from whole-genome amplification products. The prevalence of epibiotic bacteria was higher in cultures and dependent of the host species. Culture approaches demonstrated that both diatoms carry distinct bacterial communities in short-and long-term associations. Bacterial epibonts, commonly associated with phytoplankton, were repeatedly isolated from cells of diatom collection cultures but were not recovered from environmental cells. Our results suggest that in controlled laboratory culture conditions bacterial-diatom and bacterial-bacterial interactions select for a simplified, but specific, epibiotic microbiota shaped and adapted for long-term associations.