Vitex L. is the largest genus of the Lamiaceae family, and most of its species are used in the traditional medicinal systems of different countries. A systematic review was conducted, according to the PRISMA methodology, to determine the potential of Vitex plants as sources of antimicrobial agents, resulting in 2610 scientific publications from which 141 articles were selected. Data analysis confirmed that Vitex species are used in traditional medicine for symptoms of possible infectious diseases. Conducted studies showed that these medicinal plants exhibited in vitro antimicrobial activity against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Vitex agnus-castus L. and Vitex negundo L. have been the most studied species, not only against bacterial strains but also against fungi such as Aspergillus niger and Candida albicans, viruses such as HIV-1, and parasites such as Plasmodium falciparum. Natural products like agnucastoside, negundol, negundoside, and vitegnoside have been identified in Vitex extracts and their antimicrobial activity against a wide range of microbial strains has been determined. Negundoside showed significant antimicrobial activity against Staphylococcus aureus (MIC 12.5 µg/mL). Our results show that Vitex species are potential sources of new natural antimicrobial agents. However, further experimental studies need to be conducted.