Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative bacterial pathogen associated with severe periodontitis and non-oral diseases. Clinical isolates of Aa display a rough (R) colony phenotype with strong adherent properties. Upon prolonged culturing, non-adherent strains with a smooth (S) colony phenotype emerge. To date, most virulence studies on Aa have been performed with S strains of Aa, whereas the virulence of clinical R isolates received relatively little attention. Since the extracellular proteome is the main bacterial reservoir of virulence factors, the present study was aimed at a comparative analysis of this sub-proteome fraction for a collection of R isolates and derivative S strains, in order to link particular proteins to the virulence of Aa with serotype b. To assess the bacterial virulence, we applied different infection models based on larvae of the greater wax moth Galleria mellonella, a human salivary gland-derived epithelial cell line, and freshly isolated neutrophils from healthy human volunteers. A total number of 351 extracellular Aa proteins was identified by mass spectrometry, with the S strains consistently showing more extracellular proteins than their parental R isolates. A total of 50 known extracellular virulence factors was identified, of which 15 were expressed by all investigated bacteria. Importantly, the comparison of differences in exoproteome composition and virulence highlights critical roles of 10 extracellular proteins in the different infection models. Altogether, our present study provides novel cues for understanding the virulence of Aa, and for development of potential preventive or therapeutic avenues to neutralize this important oral pathogen.