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Molds are ubiquitously distributed in nature and their spores can be found in the atmosphere even at high altitudes. The difficulty of controlling these undesirable molds, as well as the growing interest of the consumers in natural products, have been forcing the industry to find new alternatives for food preservation. The modern trends in nutrition suggest the limitation of synthetic food additives or substitution with natural ones. Aromatic herbs are probably the most important source of natural antimicrobial agents. Origanum heracleoticum L. essential oil has been known as an interesting source of antimicrobial compounds to be applied in food preservation. In the this work, we have investigated the effect of essential oil obtained from O. heracleoticum on growth of six isolates of Penicillium aurantiogriseum and four isolates of Penicillium chrysogenum isolated from meat plant for traditional Petrovacka sausage (Petrovská klobása) production. The findings reveal that the essential oil of O. heracleoticum provides inhibition of all of fungal isolates tested. O. heracleoticum L. essential oil exhibited higher antifungal activity against the isolates of P. chrysogenum than the isolates of P. aurantiogriseum. O. heracleoticum essential oil showed a MIC value ranging from 25 to 100 μL/mL. The fungi cultivated in the medium with higher concentration of essential oil showed certain morphological changes. The alterations included lack of sporulation and loss of pigmentation.