Antimicrobial resistance is a global threat, leading to an alarming increase in the prevalence of bacterial infections that can no longer be treated with available antibiotics. The World Health Organization estimates that by 2050 up to 10 million deaths per year could be associated with antimicrobial resistance, which would equal the annual number of cancer deaths worldwide. To overcome this emerging crisis, novel anti-bacterial compounds are urgently needed. There are two possible approaches in the fight against bacterial infections: a) targeting structures within bacterial cells, similar to existing antibiotics; and/or b) targeting virulence factors rather than bacterial growth. Here, for the first time, we provide a comprehensive overview of the key steps in the evaluation of potential new anti-bacterial and/or anti-virulence compounds. The methods described in this review include: a) in silico methods for the evaluation of novel compounds; b) anti-bacterial assays (MIC, MBC, Time-kill); b) anti-virulence assays (anti-biofilm, anti-quorum sensing, anti-adhesion); and c) evaluation of safety aspects (cytotoxicity assay and Ames test). Overall, we provide a detailed description of the methods that are an essential tool for chemists, computational chemists, microbiologists, and toxicologists in the evaluation of potential novel antimicrobial compounds. These methods are cost-effective and have high predictive value. They are widely used in preclinical studies to identify new molecular candidates, for further investigation in animal and human trials.