Abstract According to the CDC, Periodontitis affects 47.2% of individuals aged 30 and over. By the age of 65, the number of people impacted by periodontitis jumps to 70.1%. Dysbiosis, shifts in biofilm composition leading to imbalances in microbial composition are precursors of widespread human diseases such as tooth decay (dental caries) and periodontitis. How shifts in biofilm spatial structure impact the progression from health to disease is unknown. We have developed an in vitro dental plaque culture system in which plaque samples obtained from healthy donors via flossing are used to seed complex communities. Quantitative analysis by Fluorescence in situ Hybridization (FISH), confocal microscopy, and spectral imaging demonstrated that model biofilms are highly diverse, spatially structured, and unexpectedly heterogeneous in spatial structure. We observed that the dental plaque inocula from healthy donors were composed of single cells and large multispecies coaggregates. We hypothesize that early stochastic events mediated by these multispecies coaggregates lead to highly spatially structured and heterogeneous biofilms depending on where these interacting communities land on nascent surfaces. To test our hypothesis, we developed a protocol for disrupting plaque coaggregates. We observed that biofilms derived from dental plaque inocula in which aggregates were disrupted before seeding in vitro cultures resulted in biofilms with decreased diversity and increased spatial homogeneity. Biofilms derived de-aggregated plaque inocula lacked many gram-negative, obligate anaerobe community members. Our results demonstrate a previously unknown role multispecies aggregates in structuring oral biofilms and may have clinical importance in the mouth.