Abstract: Objectives: The purpose of this study was to evaluate the antimicrobial effect of Rubia cordifolia root water and methanol extracts on various microorganisms using the agar well diffusion method and to evaluate the variants between the two techniques used in growing bacteria in the agar well diffusion method; the pour plate technique and the spreading technique. Materials and Methods: Water and methanol extracts of R. cordifolia roots were prepared and their antimicrobial effects on various microorganisms were evaluated by the agar well diffusion method. In agar well diffusion method, microorganisms were grown by either pour plate or spreading techniques. Statistical differences between the inhibition zones diameters resulted by using the spreading and the pour plate techniques were measured by ANOVA. Results: Rubia cordifolia root methanol extract showed antibacterial activity against all the three Gram-positive bacteria used in this study and four Gram-negative bacteria and showed antifungal activity against Candida albicans. Interestingly R. cordifolia root methanol extract showed activity against 3 out of 4 bacteria which showed resistance to all antibiotics used in this study. Rubia cordifolia root water extract showed antibacterial activity only against two Gram-positive bacteria. The study found that in agar well diffusion method using pour plate technique created significantly wider inhibition zone compared to the inhibition zone created by the spreading technique at similar concentrations of the extract. Conclusion: Rubia cordifolia root extracts showed antimicrobial effects. Using pour plate technique in agar well diffusion method is more sensitive in showing antimicrobial effectiveness than using spreading technique.