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<i>Blastocystis</i> is a common intestinal protist distributed worldwide, infecting humans and a wide range of domestic and wild animals. It exhibits an extensive genetic diversity and, so far, 25 distinct small subunit ribosomal RNA (<i>SSU</i> rRNA) lineages termed subtypes (STs)) have been characterized; among them, 12 have thus far been reported in humans. The aims of the present study were to detect and genetically characterize <i>Blastocystis</i> sp. in synantropic animals to improve our current knowledge on the distribution and zoonotic transmission of <i>Blastocystis</i> STs in Italy. Samples were collected from <i>N</i> = 193 farmed animals and submitted to DNA extraction and PCR amplification of the <i>SSU</i> rRNA. <i>Blastocystis</i> was detected in 60 samples (31.08%) and successfully subtyped. Phylogenetic analysis evidenced that the isolates from fallow deer, goats, and pigs (<i>N</i> = 9) clustered within the ST5; those from pheasants (<i>N</i> = 2) in the ST6; those from chickens (<i>N</i> = 8) in the ST7; those from sheep (<i>N</i> = 6) in the ST10; and those from water buffaloes (<i>N</i> = 9) in the ST14 clade. The comparison between the present isolates from animals and those previously detected in humans in Italy suggested the animal-to-human <i>spillover</i> for ST6 and ST7. The present study represents the widest <i>Blastocystis</i> survey performed thus far in farmed animals in Italy. Further epidemiological studies using molecular approaches are required to determine the occurrence and distribution of <i>Blastocystis</i> STs in other potential animal reservoirs in Italy and to define the pathways of zoonotic transmission.