Background Antibiotic resistance is a major contributing factor to global morbidity and mortality and is associated with inappropriate medication use. However, the level of antibiotic consumption and knowledge about antibiotic resistance in Ghana is inadequately quantified. Our study identifies strategies for improved stewardship of antibiotics to prevent the proliferation of resistant pathogens by assessing the level of antibiotic knowledge, attitudes, and consumption behaviors by region, gender, age, and education in rural and urban Ghana. Methods A cross-sectional study was conducted in 12 communities in the urban Greater Accra and rural Upper West regions of Ghana. A questionnaire survey was administered to 400 individuals aged 18 years and older in selected locations during September-October 2018 to collect data on individual knowledge, attitudes, and practices concerning antibiotics and antibiotic resistance. Multivariate analysis was used to investigate the association between demographic characteristics and knowledge, attitudes, and related behaviors. Results Over 30% (125/400) had not received a doctor’s prescription during their last illness. Seventy percent (278/400) had taken at least one antibiotic in the year prior to the survey. The top five frequently used antibiotics were Amoxicillin, Amoxicillin-clavulanic acid, Ampicillin, Ciprofloxacin, and Metronidazole. Women and older adults had higher knowledge compared to their respective counterparts (p<0.01). Furthermore, prudent antibiotic use was significantly more prevalent in women than men (p<0.05). Although no regional differences were found in overall knowledge, compared to urban residents, individuals residing in rural settings exhibited higher knowledge about the ineffectiveness of antibiotics for viruses like the cold and HIV/AIDS (p<0.001). Two hundred and fifty-two (63%) respondents were unaware of antibiotic resistance. There was generally a low level of self-efficacy among participants regarding their role in preserving the effectiveness of antibiotics. Conclusion Antibiotic knowledge, attitudes, and use varied significantly across demographics, suggesting a context-specific approach to developing effective community interventions.