Anise (Pimpinella anisum L.) essential oils are intensely investigated worldwide for the beneficial properties, due to the specific bioactive compound’s structure. (1) Background: This study characterized the structure of the Pimpinella anisum essential oil and evaluated its antimicrobial properties. (2) Methods: An evaluation of the antibacterial and antifungal activity targeted strains of Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), Streptococcus pyogenes (ATCC 19615), and levure Candida albicans (ATCC 10231). Gas chromatography coupled with mass spectrometry (GC/MS) was used for structure identification, and the optical density mass loss was applied for the analysis of different dilutions of aniseed essential oils antimicrobial activity. (3) Results: A total of 13 compounds were identified, of which trans-anethole was in the highest proportion (72.49%), followed by limonene (10.01%), anisole (5%), and α-pinene (3.26%). The results obtained and statistically analyzed, utilizing one-way ANOVA with Bonferroni’s multiple comparison test, indicated the antimicrobial activity (p < 0.001) of anise essential oil. (4) Conclusion: Anise essential oil is a promising phyto-remedy with important antimicrobial activity against both Gram-positive and Gram-negative pathogens. Inhibition high percentages were found for the p. aeruginosa and S. aureus strains, but also excellent antifungal activity against C. albicans was ascertained.