In recent years, the use of natural bioactives in food, pharmaceutical and cosmetic industries has emerged as a global formulation development trend. Although natural bioactives exhibit promising properties, they are also associated with chemical instability or poor aqueous solubility. One such bioactive with beneficial functionalities but limited industrial applicability within industry is propolis. The purpose of this study was to investigate means to enable enhancement to the antibacterial activity of propolis-based aqueous formulations. Dry propolis was firstly extracted from crude material and the effect of common carrier phases used for dissolution of propolis for antibacterial assays was investigated. Consequently, the extract was formulated into propolis sub-micron aqueous dispersions via direct ultrasonication. Processing time was varied, and all formed particles were characterised immediately after production in terms of size, polydispersity and zeta potential, and then again after a month-long storage period. When tested on E. coli cells, 15% propolis dispersions caused a bactericidal effect, which was sonication time and time of exposure dependent. Particles formed at the shortest sonication period (4 min) resulted in higher cell injury while those processed the longest (10 min) caused greater cell death and with AFM imaging, cell membrane alterations were confirmed. Chemically, for whole dispersions and carrier phases alone, free radical scavenging activity and total phenol content were slightly enhanced at longer sonication times. Overall, the present work suggests that formulating propolis extract sub-micron aqueous dispersions via sonication enhances their antibacterial performance via a synergistic effect involving both their carrier and dispersed phases.