Aggregatibacter aphrophilus is a ubiquitous colonizer of the human oral cavity and is known to cause bone and joint infections that are presumed to be seeded by hematogenous spread. We recently isolated A. aphrophilus from a lower leg abscess and two cases of pediatric osteomyelitis, and compared them to a strain isolated from the oral cavity of a healthy individual. All the isolates demonstrated rough-appearing colonies and extremely tenacious biofilms on solid abiotic surfaces. No single biofilm disrupting agent (sodium metaperiodate [NaPer], dispersin B, DNAse, proteinase K) was able to disperse the biofilm, but a combination of NaPer or dispersin B followed by proteinase K, suggesting that protein and polysaccharide components are critical. In the close relative, A. actinomycetemcomitans, this biofilm phenotype is mediated by Flp pili produced by the tight adherence (tad) locus. Whole genome analysis revealed that the clinical isolates are not clonal and had intact tad loci. Using transposon mutagenesis (IS903ɸkan) we show that disruption of the genes of the tad locus abrogates the strong biofilm-forming capacity of A. aphrophilus. Transposon insertions were identified in eight genes; flp-1, rcpA, rcpC, tadZ, tadA, tadB, tadC, and tadD. All mutants were defective in rough colony formation, autoaggregation, adherence and production of Flp pili. Complementation using wild-type genes expressed in trans from an IncQ plasmid restored all of the tight adherence-associated phenotypes showing that the tad genes are functional and required for adherence in A. aphrophilus. Tad-minus strains also were attenuated in adherence to bone in an ex vivo model. In addition to a potential role in oral colonization, we propose that the tad genes contribute to the colonization and pathogenesis of osteoarticular infections by facilitating binding to bone and associated tissues.