The aim was to determine the association between healthcare workers' (HCWs) country of birth and their knowledge of appropriate use of antibiotics, and whether the association changed after an educational intervention. Older residents in nursing homes have been recognized to receive excessively antibiotic treatments. HCWs often represent an important link between the older resident and the general practitioner prescribing the antibiotics, thus their knowledge of appropriate use of antibiotics is important. This study was conducted as a prospective pre-post study. Totally, 312 HCWs from 7 nursing homes in Denmark were included. For statistical analyses, χ2 test and a linear mixed regression model were applied. Native HCWs were more likely to have a higher percentage of correct responses to single statements related to knowledge of appropriate use of antibiotics. Native HCWs had a significantly higher knowledge-of-antibiotic score compared to foreign HCWs (-7.53, P < 0.01). This association remained significant after adjusting for relevant covariates (-5.64, P < 0.01). Native HCWs' mean change in knowledge-of-antibiotic score after the intervention did not differ from the foreign HCWs' mean change in knowledge-of-antibiotic score. Our findings indicate that HCWs born outside Denmark reveal a lower knowledge-of-antibiotic score than HCWs born in Denmark despite comparable educational backgrounds. All participants increased their knowledge from baseline to follow-up. Our findings also indicate that an educational seminar cannot equalize the difference in knowledge between native and foreign HCWs. Studies with larger sample size and a more detailed measurement of cultural identity should investigate this association further.