Antimicrobial resistance (AMR) is a major public health threat, reducing treatment options for infected patients. AMR is promoted by a lack of access to rapid antibiotic susceptibility tests (ASTs). Accelerated ASTs can identify effective antibiotics for treatment in a timely and informed manner. We describe a rapid growth-independent phenotypic AST that uses a nanomotion technology platform to measure bacterial vibrations. Machine learning techniques are applied to analyze a large dataset encompassing 2762 individual nanomotion recordings from 1180 spiked positive blood culture samples covering 364 Escherichia coli and Klebsiella pneumoniae isolates exposed to cephalosporins and fluoroquinolones. The training performances of the different classification models achieve between 90.5 and 100% accuracy. Independent testing of the AST on 223 strains, including in clinical setting, correctly predict susceptibility and resistance with accuracies between 89.5% and 98.9%. The study shows the potential of this nanomotion platform for future bacterial phenotype delineation.|Sturm et. al developed a 2 to 4 h antibiotic susceptibility test based on bacterial vibrations. This diagnostic test applies to the most frequently found gram-negative bacteria in bloodstream infections and demonstrates its potential in contributing to faster treatment decisions.