This study aimed to test the validity of the cross-cultural adapted Nutrition Literacy Assessment Instrument for Brazilians (NLit-Br). An observational cross-sectional study was performed in chronic disease clinics from the Brazilian Public Health System in two phases: (1) linguistic and cultural adaptation and (2) validity testing. Six registered dietitians and thirty adult patients diagnosed with at least one chronic disease participated in the study using the nutrition literacy assessment instrument (NLit-Br) and the short assessment of health literacy for Portuguese-speaking adults (SAHLPA-18). Sample descriptive variables: age, sex, race, income, education, and occupation. To adapt the instrument to the Brazilian Portuguese and Brazilian culture, we tested cognitive interviewing and the Scale Content Validity Index (S-CVI) with a group of dietitians and patients. To test the tool’s validity, health literacy (SAHLPA-18) was used as a construct that presents similarities and differences with nutrition literacy (NLit-Br). The correlation of NLit-Br and the SAHLPA-18 was tested (Spearman’s Rho). Internal consistency was measured by Kuder–Richardson Formula 20 (KR-20). The NLit-Br content validity (S-CVI = 0.85) and internal consistency (KR-20 = 0.868) were confirmed. Additionally, NLit-Br presented a significant and robust correlation with SAHLPA-18 (r = 0.665, p < 0.001). Therefore, the NLit-Br was considered a linguistic, cultural, and valid instrument to measure Brazilian’s nutrition literacy.