Blastocystis hominis is probably the most common protozoan found in the human gut worldwide. In Taiwan, the prevalence of B. hominis infection is yet to be determined but is expected to be relatively higher among foreign workers. No data is available on the prevalence of B. hominis infection in long-term care facilities in Taiwan. This study included 713 subjects (552 residents and 161 care workers) from ten long-term care facilities in Taiwan who completed stool microscopic examinations with Merthiolate-iodine-formalin stain technique. The prevalence rate of blastocystosis was the highest among foreign and domestic care workers followed by residents (12.2%, 4.6%, and 2.7%, respectively). Older age (p = 0.04) and lower educational level (p = 0.008) were significantly associated with blastocystosis among care workers. Among residents, B. hominis infection was negatively associated with prolonged use of antibiotics within 3 months prior to examination (p = 0.05) and positively associated with tracheostomy in-place (p = 0.028). In conclusion, B. hominis infection was the most prevalent intestinal parasitic infection among both care workers and residents of long-term care facilities in Taiwan. Use of antibiotics was negatively associated with B. hominis infection among residents. Additionally, appropriate preventive measures should be implemented to older care workers with lesser educational attainment in order to reduce the risk of blastocystosis infection.