Due to the continued emergence of resistance and a lack of new and promising antibiotics, bacterial infection has become a major public threat. High-throughput screening (HTS) allows rapid screening of a large collection of molecules for bioactivity testing and holds promise in antibacterial drug discovery. More than 50% of the antibiotics that are currently available on the market are derived from natural products. However, with the easily discoverable antibiotics being found, finding new antibiotics from natural sources has seen limited success. Finding new natural sources for antibacterial activity testing has also proven to be challenging. In addition to exploring new sources of natural products and synthetic biology, omics technology helped to study the biosynthetic machinery of existing natural sources enabling the construction of unnatural synthesizers of bioactive molecules and the identification of molecular targets of antibacterial agents. On the other hand, newer and smarter strategies have been continuously pursued to screen synthetic molecule libraries for new antibiotics and new druggable targets. Biomimetic conditions are explored to mimic the real infection model to better study the ligand–target interaction to enable the designing of more effective antibacterial drugs. This narrative review describes various traditional and contemporaneous approaches of high-throughput screening of natural products and synthetic molecule libraries for antibacterial drug discovery. It further discusses critical factors for HTS assay design, makes a general recommendation, and discusses possible alternatives to traditional HTS of natural products and synthetic molecule libraries for antibacterial drug discovery.