Proper diagnosis of Blastocystis hominis in not performed routinely in medical laboratories of developing countries; consequently clinical significance of this common intestinal protozoon is liable to remain unsettled. Food-handlers are more prone to get and transmit this feco-oral infection. This work compared the sensitivity of direct diagnostic methods to detect B. hominis in stool, estimate the true prevalence among food-handlers in Sirte-Libya, to clarify the association between the parasite and gastrointestinal symptoms and the response to specific treatment. A total of 400 male food-handlers aged 18-50 year were included. Each was subjected to clinical questionnaire and 3 stool examinations by different methods. The results showed high prevalence of B. hominis in food-handlers (35.5%). Short- term in vitro culture (on Boeck and Derbholav's medium) was the most sensitive method for detection of B. hominis (35.5%), followed by permanent Trichrome-stained smear (27.5%); saline-sedimentation concentrated smear (21%) and direct iodine smear (14%). Of 108 cases having B. hominis alone, 68.5% were symptomatic. Diarrhea was the most frequent symptom (75.6%), followed by abdominal pain (66.2%) and flatulence (43.2%). Fecal parasite-load was significantly higher in symptomatic cases than asymptomatic; parasite and symptoms disappeared after metronidazole treatment. So, culture should be used on routine basis to detect B. hominis which should be considered pathogenic particularly when present alone in large numbers in symptomatic patients.