Blastocystis is one of the commonest enteric protozoan parasites worldwide. Despite its controversialclinical significance, frequent association with symptoms has necessitated treatment of caseswith persistent symptoms. For long time, metronidzole (MTZ) was considered as a basic drug forblastocystosis treatment, however reports of treatment failure as well as its well-known side effectshas promoted the search for more safe and effective alternatives. In vitro antiprotozoal activity ofethanolic extract of Egyptian propolis and a cysteine protease inhibitor, phenyl vinyl sulfone (PVS)on Blastocystis spp. was assessed through challenging with graded concentrations of propolis extract(125, 250, 500 & 1000μg/ml) and PVS (100, 200 and 300 μg/ml) compared to MTZ (10, 50and 100 μg/ml) and viable parasites were counted after 24, 48 and 72 hr. of incubation. Molecularsubtyping of Blastocystis spp. was done using subtype specific sequence-tagged site (STS) primers.Propolis extract inhibited the growth of Blastocystis spp. in both of the detected subtypes (ST1 andST3), which was especially observed in cultures exposed to 500 & 1000 μg/ml through all incubationperiods with the later concentration producing comparable results to MTZ. While PVS showedsignificant parasite count reduction on ST3 isolates, especially with the highest concentration, howeverthe effect on ST1 isolate was nonsignificant. These findings highlight the potential antiprotozoalactivity of propolis extract as a potent natural alternative for MTZ in treatment of blastocystosis.