Inappropriate and excessive antibiotic use fuels the development of antibiotic resistance. Determinants of antibiotic use, including knowledge and attitudes, are manifold and vary on different spatial scales. The objective of this study was to examine the associations between socio-spatially diverse urban areas and knowledge, attitudes, practices and antibiotic use within a metropolitan city. A cross-sectional survey was conducted in the general population in socio-spatially different areas in Dortmund, Germany, in February and March 2020. Three urban areas were chosen to represent diverse socio-spatial contexts (socio-spatially disadvantaged: A, intermediate: B, socio-spatially disadvantaged: C). Participants were selected via simple random sampling. The questionnaire comprised knowledge and attitude statements and questions around antibiotic use and handling practices. Differences between the areas were examined by estimating odds ratios (OR) and corresponding 95% confidence intervals by multiple logistic regression. Overall, 158 participants were included. Participants of Area C showed the lowest proportions of correct knowledge statements, indicated more often attitudes contrary to common recommendations, lower risk awareness and reported more often antibiotic use (C: 40.8%; A: 32.7%; B: 26.5%) and potential mishandling practices (C: 30.4%; A: 9.6%; B: 17.3%). The multiple logistic regression confirmed these differences. Around 42.3% (C), 33.3% (A) and 20.0% (B) of the diseases mentioned for which an antibiotic was used are mainly caused by viral pathogens. A common misconception across all areas was the perception of antibiotic resistance as an individual rather than a universal issue. This study reveals distinct differences between socio-spatially diverse urban areas within a metropolitan city, regarding knowledge, attitudes and practices around antibiotics and ABR. Our findings confirm that enhanced efforts are required to better inform the population about the adequate use and handling of antibiotics. This study emphasizes the need for future interventions to be tailored to the specific local socio-economic context.