Abstract Background and study aims We aimed to evaluate the diagnostic performance of magnifying endoscopy with narrow-band imaging (M-NBI) in superficial non-ampullary duodenal epithelial tumors (SNADETs) regarding the absence or presence of biopsy before M-NBI diagnosis. Patients and methods Clinicopathological data were retrospectively reviewed for 99 SNADETs from 99 patients who underwent endoscopic resection. The 99 tumors were divided into the non-biopsy group (32 lesions not undergoing biopsy before M-NBI examination) and the biopsy group (67 lesions undergoing biopsy before M-NBI examination). We investigated the correlation between the M-NBI diagnosis and the histopathological diagnosis of the SNADETs in both groups. Results According to the modified revised Vienna classification, 31 tumors were classified as category 3 (C3) (low-grade adenoma) and 68 as category 4/5 (C4/5) (high-grade adenoma/cancer). The accuracy, sensitivity, and specificity of preoperative M-NBI diagnoses in the non-biopsy group vs the biopsy group were 88 % (95 % confidence interval: 71.0 – 96.5) vs 66 % (51.5 – 75.5), P = 0.02; 95 % (77.2 – 99.9) vs 89 % (76.4 – 96.4), P = 0.39; and 70 % (34.8 – 93.3) vs 14 % (3.0 – 36.3), P < 0.01, respectively. Notably, in the biopsy group, the specificity of M-NBI in SNADETs was low at only 14 % because we over-diagnosed most C3 lesions as C4/5. M-NBI findings might have been compromised by the previous biopsy procedure itself. Conclusions In the non-biopsy group, the accuracy of M-NBI in SNADETs was excellent in distinguishing C4/5 lesions from C3. The M-NBI findings in SNADETs should be evaluated while carefully considering the influence of a previous biopsy.