The potential of using propolis collected from Thailand as a natural antioxidant and antimicrobial agent for food applications was investigated. The propolis extract was prepared by using different ethanol aqueous solutions, including 30%, 40%, 50% and 70%. Total phenolic content (TPC), phenolic compound and antioxidant activity of the propolis were determined using Folin–Ciocalteau method, high performance liquid chromatography (HPLC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, respectively. The antimicrobial ability was tested against Staphylococcus aureus (TISTR 118), Salmonella enteritidis (DMST 17368), Escherichia coli (TISTR 780) and Pseudomonas aeruginosa (ATCC 27853) using disc diffusion technique. The major phenolic compounds found in Thai propolis were rutin, quercetin and naringin. The TPC and DPPH radical scavenging activity increased with increasing ethanol concentration in the solvent. Propolis extract showed antimicrobial activity, in terms of inhibitory zone for S. aureus and limited growth underneath paper discs, against all tested bacteria.