The present study was conducted to isolate the most important bioactive compound from Pimpinella anisum L. fruit oil. The plant essential oil was extracted via steam distillation. Anethole was separated using a separatory funnel and detected on thin layer chromatography TLC plates in comparison with standard anethole that, served as positive control. Moreover, infrared FTIR spectrometer and High Performance Liquid Chromatography HPLC analysis were used to confirm the purity and identification of anethole. The isolated material was investigated for its antimicrobial activity against seven selected pathogenic microorganisms. The Gram-positive bacteria were Staphylococcus aureus, Bacillus cereus; and Gram-negative bacteria included; Escherichia coli, Proteus mirabils, Klebsiella pneumoniae, Pseudomonas aeruginosa; and the yeast Candida albicans. Anethole at different concentrations (1:1, 1:5, 1:10, 1:20) was active against all tested bacteria except for P. aeruginosa, and the highest inhibitory effect was observed against B. cereus (zone of inhibition: 22.3 mm) using the disc diffusion method. The minimal inhibitory concentration MIC of anethole was determined using a broth microdilution method in 96 multi-well microtiter plates. MIC values ranged from 31.2–500.0 µg/ml, and the most promising results were observed against S. aureus, B. cereus and E. coli (MIC 31.2 µg/ml) while, P. mirabilis and K. pneumonia ranked next (MIC 62.5 µg/ml). Furthermore, anethole achieved considerable antifungal activity against the yeast C. albicans (zone of inhibition range: 10.6–17.2 mm; MIC: 500.0).