This study is the first to report Blastocystis sp. in dairy cattle from Terceira Island, part of the Azores Archipelago. Blastocystis sp. is an enteric protozoan with high genetic diversity and is associated with both zoonotic and non-zoonotic subtypes (STs). The present survey aimed to fill the gap in knowledge regarding the occurrence and genetic characterization of Blastocystis sp. isolates in Holstein-Friesian cattle in this geographical area. A total of 116 stool samples were thus collected from dairy cows, and Blastocystis sp. DNA was detected using qPCR. The results revealed a 14.7% occurrence rate (17/116), with seven STs identified including ST3, ST5, ST7, ST10, ST14, ST25, and ST42, with variable frequency. Some of these STs (ST5, ST7, ST10, and ST14) are potentially zoonotic, underscoring their potential public health significance. However, the low frequency of Blastocystis sp. in this animal cohort possibly attributed to the extensive grazing system practiced on Terceira Island and the limited presence of zoonotic isolates, suggests a minimal risk of transmission from cattle to humans. This study highlights the importance of further research on the transmission dynamics of Blastocystis sp., particularly in regions with varying farming practices, to better understand its epidemiology and zoonotic potential.