Biofilms and microbial aggregates are a common mechanism for the survival of bacteria in nature. Microbial aggregates have been associated with intraradicular and extraradicular endodontic disease. One objective of this study was to assess bacteria isolated from acute endodontic infections for autoaggregation and coaggregation. Another objective was to use both a conventional visual assay and a novel fluorescent dye-staining technique to study bacterial aggregation. Sixty-two strains of bacteria were isolated from 10 clinical samples of endodontic abscesses or cellulitis. Autoaggregation was detected in 35/62 (56.45%) of the bacteria using the visual assay. Coaggregation of bacteria from each of the samples was demonstrated for 29/183 (15.85%) bacterial pairs using the visual assay and 148/183 (80.87%) using the dye-staining assay. Coaggregation was observed for each of the 15 genera assayed, especially Prevotella, Streptococcus, and Fusobacterium. The dye-staining assay using a confocal microscope was a highly sensitive method to detect aggregation of bacteria.