Hygiene of the bee products, especially for the use of people, is very important. Although many studies have been done to prevent bacterial contamination of honey and pollen, there is a lack of research on the hygiene of propolis. Although the risk of contamination by spore-forming bacteria is high, bees use propolis to clean hives. In order to determine the standard propolis quality, the sterilization process will be a matter of scientific interest in near future. Thus, this study examines the impact of sterilization process on chemical composition and antimicrobial activity of propolis. In this study, 250 grams of propolis was collected, and the sample was divided into three parts. Raw propolis (RP) was sterilized without any pretreatment, but sterilization made it sticky and hard, and it burned during the process. Both sterilized (SEEP) and non-sterilized (NSEEP) ethanol extracts of propolis samples were analyzed by GC-MS for their chemical contents. Susceptibility patterns of three bacteria (Escherichia coli, Staphyllococcus aureus, Pseudomonas aeruginosa) and one yeast (Candida albicans) strains to NSEEP and SEEP were assessed by the disc diffusion method. The MIC values were determined for both NSEEP and SEEP. The results show that NSSEP contains far more chemical components than SEEP, and its antimicrobial activity is much higher. It is easy to say that the sterilization process changes the chemical composition of propolis and leads to the loss of some substances and its antimicrobial activity. Therefore, it is not considered necessary. Instead of microbial contamination, chemical contamination constitutes a greater risk for human consumption.