Plants of the Asteraceae family are widely used all over the world as traditional medicinal taxa for the therapy of various ailments. This study aims to investigate the chemical profiles of Pluchea dioscoridis (L.) DC. essential oils (EOs) from four different natural habitats; canal habitat (CH), urban habitat (UH), agricultural habitat (AH), and wadi habitat (WH), in addition to evaluating their antimicrobial and cytotoxic effects. The EOs were extracted by hydrodistillation method and were analyzed by gas chromatography-mass spectroscopy (GC-MS) technique. Fifty-six compounds have been identified, sesquiterpenes being the major components of the EOs with 69.62% (CH), 60.68% (UH), 78.27% (AH), and 94.87% (WH). The predominant sesquiterpene were 6-epi-shyobunol (26.38%, WH), aromadendrene (16.72%, CH), Ç-muurolene (14.51%, CH), himachalol (13.48%, UH), tau-muurolol (12.5%, WH), cubebol (9.78%, WH), cubenol (9.39%, WH), and caryophyllene oxide (8.27%, WH). Moreover, the antimicrobial effects of the investigated EOs were studied against ten pathogens, including three Gram-positive bacteria (Staphylococcus aureus, Bacillus subtillis, Streptococcus mutants), three Gram-negative (Escherichia coli, Enterobacter cloacae, Proteus vulgaris), and four fungi (Candida albicans, Cryptococcus neoformas, Aspergillus fumigatus, Syncephalastrum racemosum) using standard agar disc-diffusion technique. Among the studied organisms, C. neoformas showed significant antimicrobial potency for the EOs from the four habitats. Furthermore, EOs of the four habitats exhibited potential cytotoxicity against the colon carcinoma cell line (HCT116) with LC50 of 29.4 (CH), 23.6 (UH), 18.0 (AH), and 16.1 (WH) µg/ml, respectively. No effects were observed for breast adenocarcinoma (MCF-7), and hepatocellular carcinoma (HepG2) cell lines. EOs extracted from P. dioscoridis, especially for (WH) could be used as alternatives to synthetic antifungal and anticancer agents.