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<b>Background</b>. Previous studies reported human papillomaviruses (HPVs) in middle ear tumors, whereas these viruses have been poorly investigated in chronic inflammatory middle ear diseases. We investigated HPVs in non-tumor middle ear diseases, including chronic otitis media (COM). <b>Methods</b>. COM specimens (n = 52), including chronic suppurative otitis media (CSOM) (n =38) and cholesteatoma (COMC) (n = 14), as well as normal middle ear (NME) specimens (n = 56) were analyzed. HPV sequences and DNA loads were analyzed by quantitative-PCR. HPV genotyping was performed by direct sequencing. <b>Results</b>. HPV DNA was detected in 23% (12/52) of COM and in 30.4% (17/56) of NME (<i>p</i> &gt; 0.05). Specifically, HPV DNA sequences were found in 26.3% (10/38) of CSOM and in 14.3% (2/14) of COMC (<i>p</i> &gt; 0.05). Interestingly, the HPV DNA load was higher in COMC (mean 7.47 copy/cell) than in CSOM (mean 1.02 copy/cell) and NME (mean 1.18 copy/cell) (P = 0.03 and P = 0.017 versus CSOM and NME, respectively). HPV16 and HPV18 were the main genotypes detected in COMC, CSOM and NME. <b>Conclusions</b>. These data suggest that HPV may infect the middle ear mucosa, whereas HPV-positive COMCs are associated with higher viral DNA loads as compared to NME.