Plant oils are sources of metabolites that have enormous potential for industrial applications. Herein, the chemical profile and in vitro antimicrobial activity of the essential oil (EO) from the leaves of Guatteria citriodora Ducke (Annonaceae) have been investigated for the first time. The composition of the hydrodistilled EO was analyzed using gas chromatography-mass spectrometry (GC-MS), which permitted the identification of oxygenated monoterpenes as the most highly representative class, and included citronellal (40.99%) and citronellol (14.6%) as the main compounds. The antimicrobial activity of G. citriodora EO (GcEO) was evaluated against pathogenic bacteria and phytopathogenic fungi. The experimental design was completely randomized (CRD), and used doses for each microorganism. Gram-positive strains were the most sensitive with a minimum inhibitory concentration (MIC) of 5.0 μL mL−1, while Gram-negative strains were 10.0 μL mL−1. The most potent antifungal activity was against Alternaria alternata (MIC of 1.25 μL mL−1). In addition, it fully inhibited A. alternata conidia germination at the minimum inhibitory concentration. The nucleic acid and soluble protein contents were significantly released from the conidia of A. alternata after treatment with GcEO. Using SEM (scanning electron microscopy), morphological alterations were observed in the conidia, which indicates that a lesion in the cytoplasmic membrane is one of its mechanisms of action. Overall, these results indicate that GcEO is an antimicrobial agent with potential applications in the agriculture, food, and pharmaceutical industries.