Culture-independent molecular analyses have revealed the diversity of the human oral microbiome, but around one-third of oral bacterial taxa have yet to be cultivated. In vitro biofilms established by Thompson et al. (p. 8307– 8314) from a dental plaque inoculum were found to harbor a complex bacterial community, including numerous previously uncultivated oral bacteria. A targeted approach led to the isolation of cultures of Lachnospiraceae HOT 500 from this source material. In vitro mixed-biofilm cultures can provide the first step in the isolation of novel bacteria, thereby enabling the study of their pathogenicity, antimicrobial resistance, and interactions with other bacteria and the human host.