Bacterial infections are difficult to treat due to multi-drug resistance, a global health challenge. Plant secondary metabolites, known for their broad spectrum of biological activities, offer a promising alternative. These compounds, particularly phenolics, are recognized for their antibacterial mechanisms and their potential to enhance the efficacy of conventional antibiotics. Agrimonia eupatoria, a plant from the Rosaceae family, is rich in phenolics and demonstrates a range of bioactivities, including antibacterial activity [1]. This study aimed to evaluate the antibacterial and antibiofilm properties of a 70% ethanolic–aqueous extract of the Agrimonia eupatoria herb, as well as its cytotoxicity and bacterial selectivity to assess its safety. The microdilution assay results demonstrated the extract to be significantly active against Staphylococcus aureus, Staphylococcus aureus MRSA, Listeria monocytogenes, and Shigella flexneri, with MIC values of 0.31 mg/mL for all strains, interpreted according to the MIC classification of Tamokou et al. [2 ]. Further, crystal violet staining of the biofilm biomass demonstrated the most notable antibiofilm effect against S. aureus MRSA, with a decrease in the biofilm biomass of up to 85.40%, followed by the effects against S. aureus (63.67%) and L. monocytogenes (62.24%). An increase in the biofilm biomass was observed in S. flexneri, which could be attributed to a stress response. To evaluate the safety of the extract, the selectivity index was calculated as the ratio between the cytotoxic (IC₅₀) and antimicrobial effect (MIC). The positive values obtained showed the higher selectivity of the extract towards bacteria compared to that for normal human fetal lung fibroblasts, i.e., MRC-5 cells (IC₅₀ = 1.52 mg/mL). The obtained results show that A. eupatoria possesses antibacterial and antibiofilm activity, which supports further research into its molecular mechanisms of action and potential synergistic effects with the available antibiotics. This strategy could target different sites of the bacterial cells and the pathways involved in biofilm formation, thereby improving antibacterial activity and preventing the development of resistance.