Giardia duodenalis, the most prevalent human intestinal parasite causes the disease, giardiasis. On an annual basis G. duodenalis infects ~1 billion people, of which ~280 million develop symptomatic disease. Giardiasis can be severe and chronic, causing malnutrition, stunted growth and poor cognitive development in children. Current treatment options rely on drugs with declining efficacy and side-effects. To improve the health and well-being of millions of people world-wide, new anti-Giardia drugs with different modes of action to currently used drugs are required. The Medicines for Malaria Venture's Pathogen Box, a collection of bio-active compounds specifically chosen to stimulate infectious disease drug discovery, represents an opportunity for the discovery of new anti-Giardia agents. While the anti-Giardia activity of Pathogen Box compounds has been reported, this work failed to identify known anti-Giardia controls within the compound set. It also reported the activity of compounds previously screened and shown to be inactive by others, suggesting data may be inaccurate. Given these concerns the anti-Giardia activity of Pathogen Box compounds was re-assessed in the current study. Data from this work identified thirteen compounds with anti-Giardia IC50 values ≤2 μM. Five of these compounds were reference compounds (marketed drugs with known anti-microbial activity), or analogues of compounds with previously described anti-Giardia activity. However, eight, including MMV676358 and MMV028694, which demonstrated potent sub-μM IC50s against assemblage A, B and metronidazole resistant parasites (0.3 μM and 0.9 μM respectively), may represent new leads for future drug development. Interestingly, only four of these compounds were identified in the previously reported Pathogen Box screen highlighting the importance of assay selection and design when assessing compounds for activity against infectious agents.