Background: The increase of antimicrobial resistance, mainly due to increased antibiotic use, is worrying. Preliminary evidence suggests that antibiotic use differs across ethnic groups in the Netherlands, with higher use in people of non-Dutch origin. We aimed to determine whether appropriate knowledge and use of antibiotics differ by ethnicity and whether knowledge on antibiotics is associated with antibiotic use. Methods: We performed a cross-sectional study analyzing baseline data (2011–2015) from a population-based cohort (HELIUS study), which were linked to data from a health insurance register. We included 21,617 HELIUS participants of South-Asian Surinamese, African-Surinamese, Turkish, Moroccan, Ghanaian, and Dutch origin. Fifteen thousand seven participants had available prescription data from the Achmea Health Data-base (AHD) in the year prior to their HELIUS study visit. Participants were asked five questions on antibiotic treatment during influenza-like illness, pneumonia, fever, sore throat and bronchitis, from which higher versus lower antibiotic knowledge level was determined. Number of antibiotic prescriptions in the year prior to the HELIUS study visit was used to determine antibiotic use. Results: The percentage of individuals with a higher level of antibiotic knowledge was lower among all ethnic minority groups (range 57 to 70%) compared to Dutch (80%). After correcting for baseline characteristics, including medical conditions, first-generation African Surinamese and Turkish migrants received a significantly lower number of antibiotic prescriptions compared to individuals of Dutch origin. Only second-generation Ghanaian participants received more prescriptions compared to Dutch participants (aIRR 2.09, 95%CI 1.06 to 4.12). Higher level of antibiotic knowledge was not significantly associated with the number of prescriptions (IRR 0.92, 95%CI 0.85 to 1.00). Conclusions: Levels of antibiotic knowledge varied between ethnic groups, but a lower level of antibiotic knowledge did not correspond with a higher number of antibiotic prescriptions.