Magnolia pugana (Iltis & A. Vazquez) A. Vázquez & Carvajal, an endemic Mexican species of the Magnoliaceae family, has received limited phytochemical and pharmacological attention. This study reports, for the first time, the chemical composition and in vitro bioactivities of essential oils (EOs) obtained from its leaves, flowers, and seeds. EOs were analyzed by gas chromatography–mass spectrometry (GC-MS) and flame ionization detection (GC-FID), revealing cyclocolorenone, a sesquiterpene, as the major compound (38–40%) across all plant parts. Antioxidant activity was evaluated using DPPH• and ABTS• radical scavenging assays, complemented by bioautographic thin-layer chromatography (TLC). Antibacterial activity was determined by minimum inhibitory concentration (MIC) against human pathogenic bacteria, while cytotoxicity was assessed in MCF-7 (breast) and HT-29 (colon) cancer cell lines. Seed and flower EOs exhibited the highest antioxidant activity, with IC₅₀ values of 21.5 mg/mL and 9.04 mg/mL, respectively. Strong antibacterial effects were observed against Gram-positive strains, particularly Staphylococcus epidermidis (MIC = 355.11 µg/mL) and S. aureus (MIC = 710.23 µg/mL). Leaf EO showed selective cytotoxicity toward MCF-7 cells (IC₅₀ = 27.25 µg/mL), while seed EO was most active against HT-29 cells (IC₅₀ = 54.01 µg/mL). These results suggest that M. pugana EOs, especially those from seeds, are a promising source of natural antioxidant, antimicrobial, and anticancer agents.