Abstract Antimicrobial resistance (AMR) remains a critical challenge in public health and research. AmrProfiler is a comprehensive tool with three specialized modules: identifying acquired AMR genes, resistance-associated mutations, and ribosomal RNA (rRNA) gene mutations across nearly 18 000 bacterial species. By integrating and refining data from established databases, it provides a robust framework for AMR analysis. AmrProfiler is the first to systematically report mutations in rRNA genes, offering in-depth analysis of rRNA copy numbers and mutations—key for identifying potential rRNA-associated resistance mechanisms. Its curated database includes 7600 unique AMR gene entries and over 4300 resistance-related mutations. Supporting genome assemblies in multiple formats, AmrProfiler allows users to customize detection thresholds for AMR genes, mutations, and rRNA analysis. Validation with Acinetobacter baumannii,Escherichia coli,Klebsiella pneumoniae,Staphylococcus aureus, and Staphylococcus epidermidis demonstrated that AmrProfiler accurately identified all AMR genes and mutations reported by other tools while also detecting additional resistance markers and mutations not previously recognized. By bridging AMR genotypes and phenotypes, AmrProfiler provides actionable insights that advance both research and clinical applications in AMR. AmrProfiler is freely available as an open-access web server without login at https://dianalab.e-ce.uth.gr/amrprofiler.