Giardia lamblia is one of the most common eukaryotic pathogens and is classified by the CDC as a category B agent of bioterrorism. In a departure from more traditional research focused on specific pathways or molecules, we have developed a high-throughput assay for screening libraries of small compounds for inhibitors and enhancers of trophozoite multiplication. Following a 24-h period of culture in 384-well plates in the presence of compounds, trophozoites were fixed, stained and enumerated. Quadruplicate screening of 1520 compounds from two libraries of known bioactives detected numerous inhibitory compounds. Based on a stringent cut-off of 5 standard deviations from the plate mean, 50 compounds (3.3%) were inhibitory. The activity of 3 compounds was confirmed in conventional culture. Although not meeting the threshold, one compound (indirubin) was identified as an agonist of trophozoite proliferation. Demonstrating the potential of high-throughput screening for rapidly finding new compounds which perturb G. lamblia multiplication, most of the hits identified by high-throughput screening do not appear to have been tested previously for their ability to affect G. lamblia trophozoites. High-throughput screening of bioactive compounds will open new avenues to a system-wide analysis of pathways affecting G. lamblia proliferation, and eventually to other phases of the life cycle.