The registered incidence of nonalcoholic fatty liver disease (NAFLD) in primary healthcare centers is lower than expected, suggesting a lack of awareness by primary care healthcare professionals. The implementation of educational tools for healthcare workers has been found to increase timely referral and treatment of patients. We aimed to determine healthcare workers’ knowledge of NAFLD to identify their educational needs in one marginalized region. We performed a cross-sectional survey of 261 healthcare professionals in Tlapa de Comonfort, Guerrero, Mexico from October 2019 to December 2019. We created a questionnaire that assessed domains most relevant to NAFLD knowledge. Two hundred and forty-six questionnaires were completed. Of the respondents, 38.3% were nurses and 63.4% were women. Most nurses identified NAFLD as a prevalent (89%) and preventable (93%) disease. Hypertension (33%) and obesity (84%) were recognized as risk factors. The associations between NAFLD and cancer, cirrhosis and cardiovascular disease were identified by 53%, 67% and 72% of respondents, respectively. The largest gaps were found in diagnostic workup, therapeutic approach and the current treatments. We identify modifiable knowledge gaps in NAFLD. Educational strategies for primary care workers could enhance the identification of patients with NAFLD and prevent complications.