The essential oils of the five Scrophularia species; Scrophularia chrysantha Jaub. et Spach, Scrophularia kotschyana Benth., Scrophularia olympica Boiss., Scrophularia cinerascens Boiss. and Scrophularia zuvandica Grossh. were obtained by hydrodistillation (HD) with a range of 0.10% to 0.16% yield and analazsed by GC-FID/MS. In the meantime, the volatile organic compounds (VOCs) of S. chrysantha, S. kotschyana, S. olympica, S. cinerascens and S. zuvandica were also identified with the technique of SPME GC-FID/MS and the phytochemical results were evaluated. The experimental results of this study showed that the major compounds of essential oils which were taken by the hydrodistillation were carvacrol (52.4%), 2-pentadecanone (26.7%), 2-pentadecanone (12.2%), (Z)-2-nonenal (11.2%) and carvacrol (69.1%) respectively. Isovaleraldehyde (37.1%, 27.9%), eucalyptol (13.8%), 2-ethyl furan (14.8%) and 3(Z)-hexenol (91.3%) were respectively found as the main constituents of the five Scrophularia species with SPME method. Also antimicrobial activities of the essential oils of the five Scrophularia species were screened by using agar well diffusion method. It was identified that S. olympica, S. chrysantha, and S. kotschyana, have anti-tuberculosis activity, whereas, the essential oils gained from S. zuvandica and S. cinerascens have anti-fungal activity.