Queso Fresco cheese (QFC), a type of Hispanic-style soft and fresh cheese, is a popular food in Latin-American diet. Due to its high moisture content, near neutral pH and moderate salt content, QFC provides an optimal environment for the growth of spoilage and pathogenic microorganisms. Currently, there are no effective commercial methods or technologies to reduce microorganisms in soft cheeses such as QFC. High-voltage atmospheric cold plasma (HVACP) is a novel, non-thermal technology that can be used to treat packaged food products and achieve significant reduction of pathogenic and spoilage microorganisms without compromising products’ qualities. In this study, 2 types of gases, dry air and MA50 (50% CO₂, 50% N₂), were evaluated for inactivation of Listeria innocua (LI), a non-pathogenic surrogate for Listeria monocytogenes, in QFC by HVACP treatment. Survival of LI after HVACP treatments was evaluated by enumeration on Listeria selective agar. Quality effects after HVACP treatments were analyzed for lipid oxidation, pH, and moisture content. Plasma characterization was done using optical emission spectrometry. ANOVA was used for statistical analysis of microbial and quality data. The results have shown that HVACP treatment was able to achieve a maximal of 4.9 Log₁₀ cfu/g LI reduction after dry air treatment and 1.7 Log₁₀ cfu/g reduction after MA50 treatment. Higher lipid oxidation was found in samples after dry air treatment than MA50 treatment due to the presence of O₂ in dry air. HVACP treatments have resulted an average of 2.14 mg/kg malondialdehyde (MDA) formed in dry air treated samples, and 1.07 mg/kg MDA in MA50 treated samples. Negligible changes were observed in moisture content and pH after HVACP treatments. HVACP treated samples had an average pH and moisture content of 5.5 and 48.8%, respectively, versus 5.7 and 49.4% in control samples. LI reductions were found to be dependent on the gas composition and treatment time. The results demonstrate the efficacy of HVACP treatment for LI inactivation in QFC. This study shows the potential of HVACP technology for non-thermal processing of delicate dairy products.