<p>Naturally derived compounds are powerful tools for controlling microbial infections. However, the use of these compounds to control microorganism gene expression and growth highlights a new approach to fighting against microbial infections. The current study aims to investigate the antimicrobial mode of action of combined essential oils (EOs) from <i>Citrus meyeri, Citrus paradise</i> and <i>Citrus sinensis</i> leaves by examining their effects on biofilm formation, cell membrane permeability, and bacterial lysis-related gene expression. The antibacterial mechanisms of EOs from <i>C. paradise, C. sinensis,</i> and <i>C. meyeri</i> and their combinations were investigated by evaluating their effects on microbial cell constituents at MIC and 2X MIC values, using crystal violet assays to assess biofilm formation, and performing real-time PCR to measure bacterial lysis-related gene expression. The study found that <i>Citrus</i> species EOs and their combinations had varying effectiveness in releasing cellular constituents from microbial isolates. Moreover, the results demonstrated significant inhibition of biofilm formation, ranging from 15 to 80%, with the strongest effects observed against <i>Staphylococcus aureus</i> and <i>Candida albicans</i>. <i>Citrus</i> species EOs inhibited the expression of several genes associated with bacterial energy metabolism, the tricarboxylic acid cycle, cell membrane proteins, and DNA metabolism. Certain genes were significantly upregulated, whereas others remained relatively unchanged. Overall, these findings highlight the potential of <i>Citrus</i> species as promising candidates for developing novel antimicrobial agents or adjuvants, offering valuable insights into their mechanisms of action and therapeutic potential against bacterial pathogens.</p>