[Latar belakang: Kadar Bunuh Minimal (KBM) ekstrak etanol temulawak (Curcuma xanthorriza Roxb.) terhadap Streptococcus mutans 25% dan 15% terhadap Streptococcus sanguinis single species (in vitro). Streptococcus mutans dan Streptococcus sanguinis saling berkompetisi untuk memperoleh nutrisi. Tujuan: Menganalisis efek antibakteri ekstrak etanol temulawak terhadap dual species Streptococcus in vitro. Metode: Uji antibakteri dengan metode perhitungan koloni dan kuantifikasi dengan Real-time PCR. Analisis data menggunakan Kruskal Wallis, Mann-Whitney dan Unpaired T-test. Hasil: KHM ekstrak etanol temulawak terhadap dual species Streptococcus 0,2% dan KBM 10%. Di dalam biofilm dual species Streptococcus, proporsi S.mutans lebih tinggi daripada S. sanguinis (p<0.05). Simpulan: Konsentrasi efektif ekstrak etanol temulawak sebagai antibakteri terhadap S.mutans dan S.sanguinis dalam dual species lebih rendah dari pada terhadap kedua bakteri tersebut sebagai single species. Di dalam biofilm dual species, S. sanguinis lebih sensitif terhadap ekstrak temulawak daripada S.mutans., Background: Minimal Bactericidal Concentration (MBC) of Java turmeric (Curcuma xanthorriza Roxb.) ethanol extract against Streptococcus mutans is 25% and 15% against Streptococcus sanguinis. In dental biofilm S.mutans and S.sanguinis competes each other to obtain nutrients. Objectives: Analize the antibacterial effect of Java tumeric ethanol extract (MIC and MBC) against dual species Streptococcus in vitro. Methods: Antibacteria activity of the extract was analyzed by measuring the growth of the bacteria after being exposed to the extract by counting colony formation and by quantifying the existing bacterial cell number using real-time PCR. Statistic analysis using Kruskal Wallis, Mann Whitney test and Unpaired t-test. Results: The MIC of the extract was 0,2% and the MBC was 10%. After exposure of the extract to the dual species biofilm, the growth of S.mutans was higher than S.sanguinis (p<0,05). Conclutions: Java tumeric ethanol extract is more effective against S.mutans and S.sanguinis as dual species Streptococcus than as single species. S.sanguinis is more sensitive to Java tumeric ethanol extract than S. mutans.]