The applicability of direct bioautography, the combination of planar chromatography with antimicrobial assay, is demonstrated with special emphasis on its recent developments such as BioArena and the use of genetically modified luminescent bacteria. Its methodological advancement is put into a historical perspective. In comparison with other commonly used antimicrobial susceptibility tests, the main advantage of direct bioautography resides in its simplicity, rapidity, and ability to detect separated individual matrix components exhibiting antimicrobial activity in situ. It is confirmed with examples that high-throughput direct bioautography is suitable as a biomonitoring-screening system for bioassay-guided isolation.