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Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is an important causative agent of periodontitis acting by employment of a series of virulence factors. Our aim was to evaluate the virulence traits and plasmid-mediated quinolone resistance of A. actinomycetemcomitans isolates in Iraq. A total of 1580 samples were collected from dental caries (n = 1190) and periodontitis (n = 390) among which 200 samples were positive. The antibiotic susceptibility pattern and biofilm formation were performed. The antibiotic resistance and virulence determinants were screened using polymerase chain reaction (PCR) technique. The ltx3 and ltx4 primers were used for identification of highly virulent JP2 type. A. actinomycetemcomitans was identified among dental caries (n = 114) and periodontitis (n = 86) samples. The JP2 type was identified in six periodontitis samples. Sixty (30% of) isolates were multidrug-resistant (MDR). Eighty-four (42% of) A. actinomycetemcomitans isolates formed strong biofilms and 44% of them had moderate-level biofilms. The detected virulence genes included ltxA (96%), cdtB (64%), qseB (62%), qseC (58%) and rcpA (58%). There was a significant relation between the existence of ltxA (42%, p = 0.041) and rcpA (64%, p = 0.022) genes and the biofilm formation. The JP2 genotype was identified in six adolescents with periodontitis. The rate of qnrA, qnrB, qnrC, qnrD, qnrS and aac(6')-Ib-cr plasmid-mediated quinolone resistance genes included 22%, 18%, 16%, 16%, 14% and 0%, respectively. The qnrA (66.7%) and qnrB (53.4%) genes were significantly detected higher in MDR strains. Herein, A. actinomycetemcomitans from dental caries and periodontitis had relatively high rate of resistance to β-lactams but low resistance levels to quinolones. Moreover, most of the resistant isolates carried the qnrA-S genes. A majority of them had ltxA gene, half of them contained all the virulence genes and JP2 genotype was found in six isolates from periodontitis. Furthermore, half of the isolates produced biofilms which was significantly related to the ltxA and rcpA genes. Screening of virulence genes and antibiotic resistance pattern determination contribute to the control, diagnosis and eradication of these isolates., (Copyright © 2022. Published by Elsevier Ltd.)