Propolis is a by-product of honeybee farming known for its broad therapeutic benefits around the world and is extensively used in the health food and beverage industry. Despite Australia being one of the world's megadiverse countries with rich flora and fauna, Australian propolis samples have not been explored adequately with most in vitro and in vivo studies centred on their Brazilian and Chinese counterparts. In view of this, our study was designed to investigate the chemical composition and anti-proliferative, antibacterial, antifungal, anti-inflammatory and antioxidant properties of Australian propolis (AP-1) extract to draw a comparison with Brazilian (BP-1) and Chinese propolis (CP-1) extracts. The AP-1 extract displayed significantly greater anti-proliferative activity against the MCF7 and the MDA-MB-231 metastatic breast adenocarcinoma cell lines compared to BP-1 and CP-1 (p < 0.05). Similar trends were also observed in the antibacterial (Escherichia coli and Staphylococcus aureus), anti-inflammatory (lipopolysaccharide-induced RAW264.7 macrophages) and antioxidant assays (ABTS, DPPH and CUPRAC) with AP-1 exhibiting more potent activity than BP-1 and CP-1. The ultra-high performance liquid chromatography (UPLC) coupled with quadrupole high-resolution time of flight mass spectrometry (qTOF-MS) and chemometrics implementing unsupervised PCA and supervised OPLS-DA analyses of the propolis samples from Australia, China and Brazil revealed 67 key discriminatory metabolites belonging to seven main chemical classes including flavonoids, triterpenes, acid derivatives, stilbenes, steroid derivatives, diterpenes and miscellaneous compounds. Additionally, seven common phenolic compounds were quantified in the samples. Further mechanistic studies are necessary to elucidate the modes of action of Australian propolis for its prospective use in the food, nutraceutical and pharmaceutical industries.