Background: Phyllanthus amarus (PA) is a well-known herb for its medicinal properties and widely used worldwide. PA has a significant role in Indian Ayurveda system of medicine for treating various ailments such as gonorrhea, menorrhagia, and other genital infections. Objectives: The aim of the study is to investigate antileptospiral activity and isolate the potential antileptospiral constituents of the methanol extract of PA (MPA). Materials and Methods: The primary pharmacological tests for leptospirosis were investigated by test tube dilution technique and microdilution technique. To examine the morphogenesis of experimental leptospirosis by morphologic and histological methods, albino mice were inoculated intraperitoneally with Leptospira interrogans sero group Icterohaemorrhagiae strains. Results: The activity-guided repeated fractionation for MPA through silica gel column chromatography yielded three compounds that exhibited antioxidant and in vitro, in vivo, and in silico antileptospiral activities. Based on diverse physicochemical and spectroscopic analyses (viz.,13C NMR,1H NMR, ultraviolet [UV], IR, and mass spectroscopy), the potential constituents were elucidated as 5-(3-(3,4-dimethoxybenzyl)-4-methoxy-2-(methoxymethyl)butyl)-4,7-dimethoxybenzo[d][1,3]dioxole(C1),1-(3-(3,4-dimethoxybenzyl)-4-methoxy-2-(methoxymethyl)butyl)-2,3,4,5tetramethoxybenzene(C2), and 4-(3-(3,4dimethoxybenzyl)-4-methoxy-2-(methoxymethyl)butyl)-3,6-dimethoxybenzene-1,2-diol (C3). The histopathological examinations of both kidney and liver showed promising activity with C3 at 75 and 100 μg/mL, respectively. Conclusion: The in vitro , in vivo, and in silico studies revealed that benzo methoxy class of compounds has great potential as antileptospiral agents.