The search for plant extracts with highly antimicrobial activity has been increased nowadays. This study evaluated the antimicrobial activity of Pulicaria crispa (Forsk.) Oliv., and Pulicaria undulata (L.) C.A.Mey., which have been used traditionally in Sudan as insect replants. The antimicrobial activity was evaluated against six pathogenic microorganisms, four bacteria (two Gram-positive; Bacillus subtilis and Staphylococcus aureus, two Gram-negative; Escherichia coli, and Pseudomonas aeruginosa), and two fungi (Aspergillus niger and Candida albicans) using disc diffusion method. The extraction of the crude extracts was done by maceration. The essential oils were extracted by hydro-distillation. Phytochemical screening was done using reference method. Essential oils were analyzed using Gas Chromatography Mass Spectrometry. The results indicated that all used the microorganisms were sensitive to the plants extracts. Results of the preliminary phytochemical screening showed the presence of saponins, comarins, tannins, sterols, and triterpenes, and absence of alkaloids, anthraquinones, and flavonoids. Twenty-eight and forty-five constituents were identified in P. crispa and P. undulata, essential oils, respectively. The main constituents in the essential oil of P. crispa were 1,4-ditert butylbenzene (22.81%), caryophyllene (13.19%), carvone (11.80%), and neryl(s)-2-methylbutanoate (10.33%), and for P. undulata were camphor (44.48%), and thymyl acetate (10.31%). Data from this study could be used for developing of natural bioactive agents to improve human health.