Medicinal plants are the focus of intense study, in particular whether their traditional uses are supported by real pharmacological effects, or merely based on folklore. Piper capense L.f. (Piperaceae) is used traditionally for the treatment of infectious diseases, and has the potential to be a source of novel antimicrobial compound(s). Crude solvent extracts (water, methanol, hexane and acetone) and sequentially extracted subfractions of the root-bark of P. capense were prepared, of which the hexane-soluble subfraction MsAsHs was identified as the most promising antimicrobial subfraction. Phytochemical analyses of the various extracts and subfractions using TLC with numerous mobile phases and compound selective visualising reagents revealed the presence of quinones in all of the crude solvent extracts. Alkaloids, lipids/sterols/steroids, phenolic compounds and amino acids/peptides were detected in select subfractions. Gradient reverse phase HPLC analyses using 0.1% formic acid and methanol indicated three major peaks in MsAsHs. IR spectroscopy indicated that carbonyl and hydroxyl functional groups, and aromatic characteristics were present in the major compound present in MsAsHs. Further analysis using targeted LC-MS Q-TOF and quadrupole LC-MS/MS analyses indicated an empirical formula of C11H8O3. This formula was confirmed for the isolated compound by GC-MS (HP5-MS column) that identified the compound as 5-hydroxy-2-methyl-1,4-naphthoquinone (C11H8O3 MW: 188.18) with 98% certainty using the database. Although 5-hydroxy-2-methyl-1,4-naphthoquinone (also known as plumbagin) is well-known, this is the first time that the presence of this compound is reported in the Piper genus. Antimicrobial activities of P. capense root-bark extracts and the subfractions were determined against Gram-negative and Gram-positive bacteria and a yeast strain using the disk diffusion and broth micro-dilution assays. Antimicrobial activity was observed against Gram-positive bacteria, Gramnegative bacteria as well as a yeast strain, indicating broad spectrum activity. The antimicrobial activities of the crude solvent extracts decreased in the order: acetone > methanol > hexane > water. The MsAsHs subfraction demonstrated the highest antimicrobial activity with an MIC of 29 μg/ml against both Staphylococcus aureus (ATCC 12600) and Candida albicans (ATCC 10231). HPLC eluents of this subfraction that were collected in a drop-wise fashion onto silica TLC plates and assayed by bioautography, indicated that the major compound eluting at 13.6 minutes accounted for most of the antimicrobial activity. Antioxidant activity was observed for the crude water extract, crude methanol extract, crude acetone extract, MsAsAs subfraction as well as the MsAsHs subfraction. Cytotoxicity against mammalian cells in culture was observed for the crude methanol extract, crude acetone extract, crude hexane extract and the MsAsHs subfraction when determined using C2C12 cells as well as resting and PHA stimulated lymphocytes. Stability testing of the MsAsHs subfraction revealed that the antimicrobial compounds found in this subfraction appear to be stable up to 30 days at both 25°C and 40°C when assayed against S. aureus. However, when assayed against C. albicans, there was an increase in antifungal activity from 29 μg/ml to < 7 μg/ml after 30 days at both temperatures tested. This study provides scientific support for the ethnomedical use of the rootbark of P. capense as an antimicrobial. To date, the presence of plumbagin has not been reported in any other plant in the Piper genus. Due to the significant cytotoxic activity against mammalian cells reported in the current study and the mechanism of action of plumbagin, the therapeutic potential of P. capense extracts is very limited due to non-selective cytotoxicity, despite its marked antimicrobial activity.