In South Africa, the antimicrobial activity of many indigenous plants has been investigated. In general, studies have focused on planktonic bacteria, with less attention given to bacterial biofilms. Many organisms, however, including the opportunistic pathogen Listeria monocytogenes occur more frequently as biofilms. The aim of this study was to identify and select plants that exhibit the best antilisterial activity, isolate the bioactive compounds and determine their effect on the architecture of listerial biofilms. The ethyl acetate and chloroform extracts of thirteen plants were investigated for antilisterial activity. The ethyl acetate extract of Acacia karroo and Plectranthus ecklonii showed the best antilisterial activity, exhibiting a minimum inhibitory concentration (MIC) of 3.1 mg/ml and 0.5 mg/ml, respectively. These were further selected for the identification of bioactive compounds. Column chromatographic purification of the ethyl acetate extracts of the leaves of A. karroo led to the isolation of three known pure compounds, namely epicatechin (1), β-sitosterol (2) and epigallocatechin (3). Confocal scanning laser microscopy (CSLM) showed that the biomass of the listerial biofilm was reduced when the isolated compounds were added. The aggregation of cells that were exposed to β-sitosterol and epigallocatechin was reduced from 25 μm as observed in untreated cells to b10 μm in length.