Cutaneous leishmaniasis (CL) is a major health problem in over 98 countries of the world, including Pakistan. The current treatments are associated with a number of adverse effects and availability problem of drugs. Therefore, there is an urgent need of easily available and cost effective treatments of CL- in Pakistan. The bioassay-guided fractionation and purification of crude extract of Physalis minima has led to the isolation of a new aminophysalin B (1), and eight known physalins, physalin B (2), 5ß,6ß-epoxyphysalin B (3), 5α-ethoxy-6ß-hydroxy-5,6-dihydrophysalin B (4), physalin H (5), 5ß,6ß-epoxyphysalin C (6), and physalin G (7), K (8), and D (9). It is worth noting that compound 1 is the second member of aminophysalin series, whereas compound 6 was fully characterized for the first time. The structures of compounds 1-9 were elucidated by spectroscopic techniques Whereas, the structural assignments of compounds 1 and 8 were also supported by single-crystal X-ray diffraction studies. The anti-leishmanial activity of isolated physlains 1-9 was evaluated against Leishmania major and Leishmania tropica promastigotes. Compounds 2, 3, and 5-7 (IC50 = 9.59 ± 0.27-23.76 ± 1.10 µM) showed several-fold more potent activity against L. tropca than tested drug miltefosine (IC50 = 42.75 ± 1.03 µm) and pentamidine (IC50 = 27.20 ± 0.01 µM). Whereas compounds 2, 3 and 5 (IC50 = 3.04 ± 1.12-3.76 ± 0.85 µM) were found to be potent anti-leishmanial agents against L. major, several fold more active than tested standard miltefosine (IC50 = 25.55 ± 1.03 µM) and pentamidine (IC50 = 27.20 ± 0.015 µM). Compounds 4 (IC50 = 74.65 ± 0.81 µM) and 7 (IC50 = 39.44 ± 0.65 µM) also showed potent anti-leishmanial ativity against the miltefosine-unresponsive L. tropica strain (MIL resistant) (miltefosine IC50 = 169.55 ± 0.78 µM). Molecular docking and predictive binding studies indicated that these inhibitors may act via targeting important enzymes of various metabolic pathways of the parasites.