Absent in animals and with only seven enzymatic reactions toward the synthesis of chorismate and aromatic amino acids, the shikimate pathway is a crucial target for developing antimicrobial agents and herbicides. Although this pathway has been extensively studied in microorganisms related to human health, it reveals complexities in plants, as it takes part in primary and secondary metabolism. Obtaining enzyme inhibitors is essential to circumvent the occurrence of weeds resistant to commercially available herbicides and to help control human diseases, which has challenged researchers to search for new molecules and investigate their modes of action. By applying bioinformatics tools, thousands of enzyme inhibitors of this metabolic pathway can be prospected at a low cost and in a short time. Here, we revisit how the enzymes of the shikimate pathway have been characterized and update the status of their inhibitors in microorganisms and plants. This overview can be constructive in searching for enzyme inhibitors in the academic, human health, and agro-industrial fields. © The Author(s), under exclusive licence to Springer Nature B.V. 2023.