The Polyomavirus family includes two members, BK virus (BKV) and JC virus (JCV), that naturally infect humans. These viruses are widely distributed among the population worldwide. Primary infection occurs in early childhood and remains for life clinically unapparent in immunocompetent individuals. In the context of severe immunosuppression and other predisposing factors BKV and JCV may reactivate and cause serious illnesses known as Polyomavirus-induced nephropathy and progressive multifocal leukoencephalopathy, respectively. Here we briefly examine the biological and physical characteristics and the lifecycle, namely receptor(s) interaction, mode of entry, intracellular trafficking, viral transcription and replication, and progeny assembly of these two human Polyomaviruses. We also provide an overview of the clinical manifestation of Polyomavirus-induced disorders in affected individuals and discuss the potential involvement of BKV and JCV in human cancer.