<p><i>Aerva javanica</i>&#xa0;is a significant medicinal plant with a wide geographic distribution, prevalent in many countries. The plant is employed in traditional healing practices to address a range of illnesses. This study aimed to evaluate the potential genotoxicity of aqueous <i>A. javanica</i>&#xa0;leaf extract on&#xa0;<i>Allium cepa</i>&#xa0;roots using cytogenetic and molecular assays. The effective concentration (EC₅₀) of the water-based leaf extract was determined to be 26.7&#xa0;mg/mL. Based on this value, three concentrations were selected to evaluate genotoxicity: below the EC₅₀ (13.35&#xa0;mg/mL), the EC₅₀ itself (26.7&#xa0;mg/mL), and above the EC₅₀ (40.05&#xa0;mg/mL). To establish a baseline for comparison, distilled water was utilized as a control. The inhibition of root growth was assessed, and cytotoxicity was determined by the mitotic index (MI). In addition, genotoxicity was further analyzed through the assessment of chromosomal aberrations (CA), micronuclei (MN), and Inter-Simple Sequence Repeat (ISSR) Polymerase Chain Reaction (PCR) markers as a molecular assay. Increasing the amount of&#xa0;<i>A. javanica</i>&#xa0;leaf extract resulted in a progressive shortening of root length. Additionally, at higher concentrations MI percentage decreased, indicating cytotoxicity, while CA and MN frequencies increased, suggesting genotoxicity. Furthermore, DNA damage was confirmed through ISSR-PCR marker analysis. These findings indicate that higher concentrations of <i>A. javanica</i>&#xa0;leaf extract induce genotoxic effects, highlighting the need for studies of its human use in therapeutic applications.</p> Graphical abstract <p></p>