Tropical diseases such as leishmaniosis constitute a major health concern in developing countries. Multiresistance of pathogens against classical antibiotics is a growing problem in treating infectious diseases. Thus, the search for new antibiotics is an urgent challenge. Helichrysum spp., mainly distributed in African countries, have been used in traditional and folk medicine for the treatment of several disorders including infectious diseases such as protozoal problems. In an ongoing project on the beneficial effects of Helichrysum spp., we aim to investigate and compare the anti-protozoal activities and phytochemical components of Helichrysum oligocephalum DC. and Helichrysum leucocephalum Ausfeld. Previously, we discovered pyrone and phloroglucinol derivatives as the active anti-protozoal components in Helichrysum oocephalum Boiss. [IC50 values 5.08 µg/mL and 4.01 µg/mL against Leishmania donovani and Plasmodium falciparum, respectively for dichloromethane (DCM) fraction]. As the former plants also showed similar activities to some extent [IC50 values 5.19 µg/mL and 5.42 µg/mL against L. donovani and P. falciparum respectively for DCM fraction of H. oligocephalum, IC50 value 3.8 µg/mL against P. falciparum for DCM fraction of H. leucocephalum], we decided to compare the metabolite profiles of those plants with H. oocephalum for dereplication purposes. A sensitive method coupling high-performance liquid chromatography with a photodiode-array detector (PDA) and electrospray ionization mass spectrometry (ESIMS) was optimized for separation and metabolite profiling. The LC-ESIMS metabolite profiles of the fractions from the plants were compared by applying a two-step workflow using an ACD/MS workbook suite add-in, and data clustering on an open-source web platform freeclust. The metabolites were identified by NMR and LC-PDA-ESIMS techniques. The metabolites can be categorized into major types namely flavonoids, phenolic acids, pyrone, and phloroglucinol derivatives. Phloroglucinol derivatives were mainly present in DCM fractions. DCM extract of H. oligocephalum showed the most similar profile to our previously studied extract of H. oocephalum and seems to be even much richer in pyrone and phloroglucinol derivatives. The data could emphasize the potential of Helichrysum spp. for the treatment of infectious diseases. However, more studies on the other species around the world are needed to have a much better insight.