Oregano (Origanum vulgare) essential oil (EO) has been extensively examined as an antimicrobial agent; in Mexico, the study has focused on one of the Mexican oreganos, Lippia berlandieri. Thus, the aim of this research was to qualitatively and quantitatively characterize compounds present in Poliomintha longiflora EO, another of the Mexican oreganos, and to determine its antifungal activity against several phytopathogenic molds. P. longiflora EO was obtained by steam distillation; its chemical characterization was performed by GC and GC-MS, elucidating up to twenty-eight different chemical components, including thymol (37.2%), carvacrol (24.6%), isothymolmethyl ether (8.3%), and p-cymene (8.1%). Quantitative characterization of the two major isomers determined that thymol concentration (0.332 g/g) was higher than carvacrol (0.176 g/g). Minimum inhibitory concentrations (MICs) against Penicillium expansum, Aspergillus flavus, Botrytis cinerea, or Colletotrichum gloeosporioides were determined. MICs of 1.2 and 1.4 g/L of P. longiflora EO were determined for P. expansum and A. flavus, while for B. cinerea and C. gloeosporioides MICs were 0.8 and 1.0 g/L, respectively. P. longiflora EO presented three chemical components (thymol, carvacrol, and p-cymene) that provide commercial value for oreganos from this species; furthermore, the concentration of these bioactive compounds makes P. longiflora EO a good antifungal agent.