Background Diffusion-weighted imaging (DWI) can quantitatively reflect the diffusion characteristics of tissues, providing a theoretical basis for qualitative diagnosis and quantitative analysis of a disease. Purpose To characterize testicular lesions that present as a hypointense signal on magnetic resonance imaging (MRI) T2-weighted images using DWI. Material and Methods Study participants were divided into three groups. Group A were healthy controls (n = 35), group B included patients with mumps orchitis (n = 20), and group C included patients with seminoma (n = 15). DWI sequences used b-values of 0, 1000, and 2000 s/mm2. Apparent diffusion coefficient (ADC) values between 1000 and 2000 s/mm2 were calculated by MRI postprocessing software. The Kruskal–Wallis test and receiver operating characteristic analysis were performed to evaluate how well ADC values distinguished between mumps orchitis and seminoma. Results Normal testicular tissue showed a hyperintense signal on DWI and hypointensity on the ADC map: mean ADC value was 0.77 (0.69–0.85) ± 0.08 ×10−3 mm2/s. Mumps orchitis and seminoma showed slight hyperintensity on DWI: mean ADC values were 0.85 (0.71–0.99) ± 0.15 ×10−3 mm2/s and 0.43 (0.39–0.47) ± 0.04 × 10−3 mm2/s, respectively. There were statistically significant differences in mean ADC values between normal testicular tissue and seminoma and between mumps orchitis and seminoma. The cutoff ADC value for differentiating seminoma from mumps orchitis was 0.54 × 10−3 mm2/s. The sensitivity, specificity, and Youden Index for diagnosing seminoma were 99%, 31%, and 30%, respectively. Conclusion High b-value DWI has potential utility for differentiating mumps orchitis from seminoma in the clinical setting.