Esophageal cancer is an aggressive and fatal disease classified into two distinct histologic types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). To develop novel therapeutic strategies, it is important to establish preclinical models of esophageal carcinoma. In this study, we successfully established three types of human esophageal cancer organoids (tumoroids) from surgical specimens for long-term culture. Two of the tumoroids were derived from ESCC and one from EAC, which arose from Barrett's esophagus. Whole-exome sequencing revealed that the tumoroids inherited genetic mutations from the parental tumors and patient-derived tumor xenografts closely mimicked the pathology of the original esophageal cancers. In addition to whole-exome analysis, copy number and immunohistochemical analyses demonstrated HER2 expression and amplification as well as HER3 expression and mutation in esophageal tumoroids derived from adenocarcinoma in Barrett's esophagus. HER2-targeting antibody-drug conjugates (ADCs), trastuzumab deruxtecan (T-DXd), and patritumab deruxtecan (P-DXd) effectively suppressed the viability of the tumoroids. Therefore, the establishment of esophageal tumoroids with long-term cultivability makes it possible to obtain reproducible basic data, including drug sensitivity studies, which are important for the development of personalized therapies and treatment strategies.