Irrational antibiotic use has led society to antibiotic resistance—a serious health problem worldwide. This study aimed to assess public knowledge, beliefs, and behavior concerning antibiotic use and self-medication in Lithuania. The cross-sectional survey method was processed using a validated questionnaire in different regions of Lithuania. In total, 1005 adults completed the questionnaire and were included in the study. More than half of the respondents (61.1%) had poor knowledge of antibiotics. Almost half of the respondents incorrectly identified antibiotics as being effective either against viral (26.0%) or mixed (bacterial and viral) infections (21.7%). The respondents with lower educational qualifications (OR = 2.515; 95% CI 1.464–4.319; p
= 0.001) and those from rural areas (OR = 1.765; 95% CI 1.041–2.991; p
= 0.035) were significantly less knowledgeable of antibiotics. There was no significant difference between genders, different age groups, or different parenthood status. The determined level of self–medication with antibiotics was 31.0%. The men (OR = 1.650; 95% CI 1.120–2.430; p
= 0.011), the respondents from rural areas (OR = 2.002; 95% CI 1.343–2.985; p
= 0.001), and those without children (OR = 2.428; 95% CI 1.477–3.991; p
< 0.001) were more likely to use antibiotics in self-medication. Lithuanian residents’ knowledge of antibiotics is insufficient. More information about antibiotic use should be provided by physicians and pharmacists. Self-medication with antibiotics is a serious problem in Lithuania and requires considerable attention.
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