Abstract. Fendiyanto MH, Satrio RD. 2020. Identification, aflatoxin content, and antagonistic test of spoilage fungi in bread to Aspergillus niger. Bioteknologi 17: 60-66. The major problem in shelf storage life in bread is contaminated by molds fungi and its mycotoxins. The growth of the spoilage fungi indicates the bread has structural damage. Infection from this fungus will affect the shelf life of the bread to be shorter. Contaminated bread will cause influenced human health in the future. One of the many solutions to overcome the fungi-contaminated of bread is the use of biological agents. Aspergillus niger has the ability as a biological agent in suppressing food-destroying pathogens in many foods including bread as well. However, there were few reports about the antagonistic test in bread particularly between A.niger as biological control agent to spoilage fungi. Therefore, this study aimed to analyze the growth antagonistic test of A. niger against food spoilage fungi, expecting the storage life of bread can be extended and avoid mycotoxins contamination. The antagonistic test was in the form of a test that utilizes the properties of microorganisms that grow faster than pathogens or produce antibiotic compounds. The methods used in this study included isolation and identification of fungi, water content analysis, aflatoxin test, and antagonistic test. Interestingly, the antagonist test results showed A. niger can inhibit the growth of spoilage fungi on bread. In conclusion, the species of fungus with the highest inhibition value was Hyphopichia burtonii, while the lowest was Saccharomyces cerevisiae. This funding indicates that A.niger can be used as a biological control in extending bread shelf storage in the future.