Abstract: Calendula officinalis L. (Asteraceae), commonly known as English or pot marigold, is an herbaceous plant with edible flowers. In this study, UPLC-ESI-MS/MS analysis was used for tentative identification of compounds in marigold flower methanol extract (MFE). In addition, RP-HPLC-DAD analysis was used to quantify the flavonoids hesperidin and rutin in MFE. The antileishmanial potentials of the crude extract and compounds were evaluated against Leishmania major promastigotes and amastigotes. Further, in vivo 4-day antimalarial testing of the extract and compounds was carried out at doses of 25 mg kg−1 per day using mice infected with ANKA strain of Plasmodium berghei, following standard procedure. Molecular docking studies were carried out to assess the binding mode of flavonoids against the vital targets of L. major, including pteridine reductase 1 and farnesyl diphosphate synthase enzymes. The in silico antimalarial potentials of flavonoids were evaluated against wild-type Plasmodium falciparum dihydrofolate reductase-thymidylate synthase and phosphoethanolamine methyltransferase enzymes. Twenty compounds were tentatively identified by UPLC-ESI-MS/MS analysis of MFE, of which, seven flavonoids, six saponins, three phenolic acids, three fatty acids, and a triterpene glycoside were identified. MFE phytochemical analysis revealed that hesperidin content was 36.17 mg g−1 extract, that is, 9.9-fold their content of rutin (3.65 mg g−1 extract). The method was validated to ensure reproducibility of the results. The tested samples exhibited antileishmanial potentials against L. major promastigotes, with IC50 values of 98.62, 118.86, and 104.74 ng µL −1 for hesperidin, rutin, and MFE, respectively. Likewise, hesperidin showed inhibitory potentials against L. major amastigote with an IC50 value of 108.44 ± 11.2 µM, as compared to miltefosine. The mean survival time, parasitemia, and suppression percentages showed similar results for the three samples against ANKA strain of P. berghei. The docking studies showed good binding affinities of rutin and hesperidin with numerous H-bonding and van der Waals interactions. Marigold flowers are nutraceuticals, presenting important sources of bioactive flavonoids with potential against neglected tropical diseases.