Background: Petalostigma pubescens and Petalostigma triloculare were common components of pharmacopeia's of multiple Australian Aboriginal tribal groupings which traditionally inhabited the areas in which they grow. Among these groups, they had a myriad of medicinal uses in treating a wide variety of bacterial, fungal and viral infections. This study was undertaken to test P. pubescens and P. triloculare leaf and fruit extracts for the ability to inhibit bacterial and viral growth and thus validate Australian Aboriginal usage of these plants in treating bacterial and fungal diseases. Materials and Methods: P. pubescens, and P. triloculare leaves and fruit were extracted and tested for antimicrobial, antiviral activity and toxicity. The bioactive extracts were further examined by RP-HPLC and GC-MS to identify the component compounds. Results: The methanol, water and ethyl acetate leaf and fruit extracts of displayed potent antibacterial activity. The methanol and ethyl acetate extracts displayed the broadest specificity, inhibiting the growth of 10 of the 14 bacteria tested (71%) for the leaf extract and 9 of the 14 bacteria tested (64%) for the fruit extracts. The water extracts also had broad spectrum antibacterial activity, inhibiting the growth of 8 (57%) and 7 (50%) of the 14 bacteria tested, respectively. All antibacterial extracts were approximately equally effective against Gram-positive and Gram-negative bacteria, inhibiting the growth of 50-75% of the bacteria tested. The methanol, water and ethyl acetate extracts also displayed antiviral activity in the MS2 plaque reduction assay. The methanol and water extracts inhibited 26.6-49.0% and 85.4-97.2% of MS2 plaque formation, respectively, with the fruit extracts being more potent inhibitors. All ethyl acetate extracts inhibited 100% of MS2 plaque formation. All extracts were also non-toxic or of low toxicity. Analysis of these extracts by RP-HPLC showed that the P. triloculare ethyl acetate fruit extract was the least complex of the bioactive extracts. Subsequent analysis of this extract by GC-MS revealed that it contained 9 main compounds: acetic acid; 2,2-dimethoxybutane; 4-methyl-1,3-dioxane; decane; unadecane; 2-furanmethanol; 1,2-benzenediol; 1,2,3-benzenetriol; and benzoic acid. Conclusion: These studies validate Australian Aboriginal therapeutic usage of Petalostigma species and indicate their medicinal potential.