This chapter focuses on methods of polyphenol isolation and on the antibacterial activity of different polyphenols found in herbs, spices, fruits and vegetables. Polyphenols are secondary metabolites which protect plants from different pathogens, such as viruses, bacteria, fungi, insects, and herbivores. Currently, about 9000 polyphenols found in more than 480 plants are known. Their amount fluctuates across different species and varieties. This chapter describes conventional and novel methods for extraction, the influence of the type of solvents, solvent concentration and temperature on the yield. The highest yield is obtained at 70% of methanol and ethanol, and at 90% of acetone. Extraction at 80°C leads to higher amounts of polyphenols than extraction at 100°C. Polyphenols are usually metabolized in the human liver but can also remain unaffected as they pass through the gastrointestinal tract. The main location for their uptake is the colon. They exhibit a wide range of antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus epidermidis, Klebsiella pneumoniae, E. coli, Listeria monocytogenes, Acinetobacter sp., Proteus sp., Micrococcus sp., and Bacillus sp. All these plants, rich in antimicrobial polyphenols, represent a promising and powerful source of highly effective novel antibacterial substances in the current era of ubiquitous antibiotic resistance.