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• Essential oil of Siparuna guianensis showed antimicrobial activity. • Bacteria exposed to S. guinanensis essential oil showed higher membrane permeability. • Essential oil S. guianensis showed no destructive interactions with human cells. • Germacrene B was the compound with the best interaction with the polymerases. The increasing prevalence of resistance to conventional antibiotics in pathogenic bacteria has demanded faster development of novel sources of antibacterial agents. In this context, biological activities shown by natural compounds have received particular attention. One of the alleged backslashes for these alternative bactericidal tools is the current knowledge gap regarding their action mechanisms. Here, we evaluated the activity of essential oil extracted of Negramina, Siparuna guianensis , plants against pathogenic bacteria Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa and Streptococcus pyogenes. We evaluated cytotoxic effects on bacterial cells and the action mechanisms of the essential oil. The gas chromatography analysis revealed that β-Myrcene (39.68%), epicurzerenone (18.16%) and germacrenes D (14.34%) and B (2.93%) are the major components of the S. guianensis essential oil. Interestingly, the essential oil exhibited toxicity against all pathogenic bacteria without affecting the human monocytic THP-1 cell line. This antibacterial activity resulted from strong bacterial growth inhibition and deregulation of bacterial cell wall permeability with increased nucleotides and K+ ions leakage. Furthermore, our molecular docking predictions indicated high affinity between some essential oil major components Germacrene B and active sites of bacterial DNA and RNA polymerases, which indicates possible impairments on the pathogenic bacteria cell replication processes. [ABSTRACT FROM AUTHOR]