Volatile oil of Cyperus rotundu L. rhizomes, which were collected from Kosti, White Nile Province, Sudan was obtained by hydro-distillation method. Yield percentage of oil was found to be 0.49 %. Oil was tested for its antimicrobial activity against five standard organism of bacteria and two standard organisms of fungi, it showed moderate activity against all organisms except E, coli, which was sensitive to the two concentrations used. GC MS analysis of the oil showed the presence of sixty compounds. The major compounds were cyperene (16.9 %), caryophylene oxide (8.9), β-selinene (6.6 %), α-longipinane (8.4 %), eugenol (4.7 %), aristolone (3.5 %), β-calacorene (3.3 %), α-copaene (3.2 %), trans- γ-bisabolene (3.1 %) and α-cyperone (3.0 %.).