Halophytes have long been used for medicinal purposes. However, their use was entirely empirical, with no knowledge of the bioactive compounds. The plant Tamarix aphylla L. has not drawn the deserving attention for its phytochemical and bioactive explorations, but available data expressed its needs to be attended for its potential. The Streptococcus mutans SpaP gene (cell-surface antigen) mediates the binding of these bacteria to tooth surfaces. The growing problem of antibiotic resistance triggered the research on alternative antimicrobial approaches. Our study aims to explore the activity of T. aphylla ethanolic extract against the virulence gene found in Streptococcus mutans pathogenic bacteria. Samples that were previously collected and identified in our previous work (in press) were obtained from different dental clinics and hospitals in Baghdad. Three nonbiofilm-forming bacterial isolates having multidrug resistance (MDR) for 10 antibiotics (doxycycline, ofloxacin, tetracycline, erythromycin, vancomycin, clindamycin, rifampicin, imipenem, amikacin, and cefepime) were selected to examine the potential of the T. aphylla ethanolic extract. The ethanolic extract showed high antimicrobial activity against MDR. Minimum inhibition concentration (MIC) for the extract was 17.5 mg/ml, while minimum bactericidal concentration (MBC) was 35 mg/ml. The phytochemical compounds present in the ethanolic extract were determined by using high-performance liquid chromatography (HPLC) which revealed that the leaves contain thirteen different alkaloids, twelve flavonoids, and four vitamins. The extract strongly inhibited a virulence property, the adherence of S. mutans which reduced during critical growth phases. The one-step RT-PCR method was used to study the SpaP gene expression of bacterial isolates which significantly reduced. In conclusion, extraction of T. aphylla leaves showed an antimicrobial effect against MDR S. mutans. The identified phytochemicals in the T. aphylla extract are reported to be biologically important and need further investigation to develop safe and cheap drugs. CI - Copyright (c) 2023 Sahira A. Aljubouri and Khalid H. Alobaidi.