Keratoacanthomas (KA) and Spitz naevus (SN) are both lesions with unknown aetiology; therefore, the possibility of a viral involvement, more specifically the involvement of human polyomaviruses (HPyV), was investigated. In total, 22 cases of KA and 25 cases of SN were tested for the presence of HPyVs. DNA was extracted and amplified by multiplex PCR and thereafter tested with a multiplex bead-based assay for HPyVs (BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, TSPyV, HPyV6, 7 and 9) and two primate viruses (SV40 and LPyV). HPyV DNA was found in 20 of the 47 lesions. There was no significant difference in HPyV DNA detection frequency between patients diagnosed with KA and patients diagnosed with SN, nor any over-representation of a specific HPyV type in any of the two patient categories. In conclusion, evidence for a specific aetiological role of any of the above tested HPyVs in either KA or SN was not disclosed.