The aim of this study was to investigate the antimicrobial activity against Staphylococcus aureus, Cutibacterium acnes, Escherichia coli, and Candida albicans of the ethanol extract and the n-hexane and ethyl acetate extract of the stingless bee Tetragonula sp. propolis. Additionally, the study also identified the stingless bee species based on morphological characteristics and Cytochrome c oxidase subunit I (COI) gene sequence. The antimicrobial activity of three stingless bee propolis extracts showed resistance against all three bacterial S. aureus, C. acnes, and E. coli. At a concentration of 1000 μg/mL, the ethanol extract exhibited the highest resistance, with C. acnes showing a zone of inhibition of 13.33 mm, while the inhibition zones were 11.33 mm for S. aureus and 10.33 mm for E. coli. The Minimum Inhibitory Concentration (MIC) for the three bacterial S. aureus, C. acnes, and E. coli were found to be 30, 50, and 50 μg/mL, respectively. The study identified the presence of polyphenols, flavonoids, aglycones, phenolics, and ketones quantified the total phenolic and flavonoid content, assessed the antibacterial activity of the propolis extract against the three pathogenic bacteria, and provided the foundation for further research.