Essential oils (EOs) are some of the most important natural products due to their various biological applications. In fact, many EOs explain crucial antimicrobial activities against several phyto-, food, and human diseases; hence, they are considered possible alternatives to synthetic drugs. The second volume of this Special Issue, entitled "Plant Essential Oil with Biological Activity II," includes important scientific articles that cover several aspects of the use, extraction, biochemical characterization, application, and mechanisms of action of many EOs, either crude oil or their single constituents. This Special Issue also includes crucial studies regarding the phytotoxicity and/or cytotoxicity of some plant EOs. This Special Issue's second volume addresses knowledge discrepancies in science and promotes the global development of EO applications. It includes seventeen research articles covering a broad range of subjects and uses related to the biological activity of many plant EOs.