In this study, the physicochemical, antioxidant, antibacterial properties, and the toxicity of propolis particles produced by stingless bee Heterotrigona itama found in Brunei Darussalam were investigated. Propolis particles of different sizes were extracted from raw propolis using various volume fractions of ethanol in water. Spectroscopic analyses were utilized to characterize the chemical structures, functional groups, as well as absorbance and fluorescence properties. The total antioxidant capacity of propolis particles, which was assessed using DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, was found to increase with volume fraction of ethanol. The maximum antioxidant capacity was as high as 317.65 mg ascorbic acid equivalent per gram of propolis particles. All of the propolis particles showed antibacterial activity against Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). The diameters of the inhibition zone were either significantly higher or equivalent to those of two standard antibiotics (rifampicin and streptomycin), suggesting strong antibacterial activity. The toxicity studies of propolis particles against Caenorhabditis elegans revealed that they are non-toxic after 24 h exposure. Overall findings suggest that H. itama propolis particles are not only an important source of natural antioxidants that could be beneficial for human health, but they have potentials as antimicrobial against bacteria.