This study investigated the potential of St. John’s Wort (Hypericum perforatum L.) oil as a natural alternative to nitrite in pastirma production. Three formulations were developed: samples containing nitrite and 1% St. John’s Wort oil (N-S), samples containing only 1% St. John’s Wort oil (NN-S), and a control group without nitrite or St. John’s Wort oil (NN-SS). The products were stored at +4 °C for 30 days, and analyses of moisture, ash, pH, color parameters (L*, a*, b*), and thiobarbituric acid reactive substances (TBA) were conducted on days 0, 15, and 30. Moisture content decreased progressively during storage, from an average of 41.2% at day 0 to 33.7% at day 30, with a more pronounced reduction in samples containing St. John’s Wort oil. The pH remained stable within the narrow range of 5.72–5.69, suggesting that neither nitrite nor St. John’s Wort oil induced significant acidification or microbial deterioration. Specifically, a* values were consistently higher in nitrite-treated samples, whereas b* values increased in the presence of St. John’s Wort oil, likely due to its flavonoid and pigment constituents. TBA results demonstrated lipid oxidation advanced most rapidly in the control group, reaching 1.62 mg MDA/kg at day 30. In comparison, the N-S group exhibited significantly lower values (0.95 mg MDA/kg), corresponding to approximately 41% reduced oxidation. Overall, the findings indicate that while St. John’s Wort oil alone cannot fully substitute for nitrite, it contributes to moisture reduction, mineral enrichment, pH stabilization, and retardation of oxidative deterioration.