The defining property of an antibacterial agent is its ability to selectively interfere with bacterial growth and/or survival. Consequently, a considerable and crucial part of the preclinical evaluation of any novel antibacterial drug involves judging and characterising its effects on bacteria in vitro. These critical stages in drug development are sometimes made to appear somewhat trivial, sandwiched as they are between the highly demanding antibacterial discovery process and the formidable task of demonstrating safety and efficacy in vivo. However, careful biological evaluation in vitro is key to quantifying and understanding the basis of the antibacterial activity, providing preliminary indications and evaluations of therapeutic potential, assessing the likelihood for the development of bacterial resistance, guiding chemical refinement and assisting subsequent stages of the appraisal of any new antibacterial drug. This review covers concepts in, and strategies for, the in vitro microbiological and molecular evaluation of antibacterial drug candidates.