Background/Aims@#Because gastrointestinal tract is not sterile, primary culture has contaminationrisk despite of massive washing with antimicrobial media. Microbial contamination can play a key role in initial failure during biopsy-derived primary tumorculture. @*Methods@#Tumortissue was acquired from esophageal and gastric tumors using endoscopic biopsy. Three-dimensional cultures were performed, and separated spheroids were cultured in media for 7 to 10 days and then transferred to Matrigel (Corning Inc.). We investigated risk factors and patterns of initial fungal contamination. @*Results@#Initial tumorcontamination was observed in 23% (7/30) of esophageal cancer and 20% (3/15) of gastric cancer samples. Two cases of bacterial contamination occurred during the establishment of culture protocol. Moderate to thick whitish plaques (p < 0.001) and food retention in lumen (p < 0.001) were risk factors for initial fungal contamination. After exclusion of high riskpatients for contamination, no fungal contamination occurred in primary organoidcultures. Fungal contamination was usually detected within 3 days after tumor preparation. However, unusual fungal contamination (GC11 and EC29) was recognized after several passages. Growing spherical shapes resembled cancerorganoids. Although they rapidly proliferated and multiple daughter spheroids appeared, the media were translucent. After several passages, yeasts and pseudohyphae were detected on the edges of the solid spherical structures and media. @*Conclusions@#Moderate to thick whitish plaques and food retention are clinical risk factors for initial fungal contamination during biopsy-derived cancerorganoidculture. Most initial fungal contamination was detected within 3 days, but it could be unusually recognized after several passages.