Background: Poor knowledge of antibiotic use drives poor antibiotic practices, but little is known about the influence of health information orientation (HIO) on knowledge of antibiotic use in the general public. Methods: We conducted a nationally-representative population-wide cross-sectional study (November 2020–January 2021), on a proportionately stratified random sample of 2004 Singapore residents aged ≥21 years. Multivariable logistic regression analysis was performed to assess the association between HIO and knowledge of antibiotic use. Results: Forty percent of respondents had low-levels of HIO (LL-HIO); they tended to be younger, not currently married, and did not have family/friends working in the healthcare sector. Respondents with LL-HIO (aOR 1.82, 95% CI 1.32–2.51, p < 0.001) were 82% more likely to have poor knowledge of antibiotic use. In particular, older adults aged ≥50 years with LL-HIO (aOR 1.81, 95% CI [1.32–2.51], p < 0.001) were much more likely to have poor knowledge than their HL-HIO counterparts. They were also less likely to use the Internet to seek health information and had poor eHealth efficacy. Conclusion: LL-HIO is independently associated with poor knowledge of antibiotic use. Educational strategies on antibiotic use should disseminate a consistent message through both online and offline platforms, involving traditional and non-traditional healthcare and non-healthcare influencers.