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Abstract This study aimed to determine the association between factors relevant to diagnostic uncertainty and physicians’ antibiotic-prescribing behaviour in primary care. A questionnaire survey was conducted on 327 physicians that measured their diagnostic ability, perceived frequency of diagnostic uncertainty, tolerance, and perceived patient tolerance of uncertainty. Physician antibiotic-prescribing behaviours were assessed based on their prescriptions (n = 207,804) of three conditions: upper respiratory tract infections (URTIs, antibiotics not recommended), acute tonsillitis (cautious use of antibiotics), and pneumonia (antibiotics recommended). A two-level logistic regression model determined the association between diagnostic uncertainty factors and physician antibiotic prescribing. Physicians perceived a higher frequency of diagnostic uncertainty resulting in higher antibiotic use for URTIs and less antibiotic use for pneumonia. Higher antibiotic use for acute tonsillitis was related to a low tolerance of uncertainty of physicians and patients. This study suggests that reducing diagnostic uncertainty and improving physician and patient uncertainty management could reduce antibiotic use.