In this study, we evaluated the efficacy of 3 commercial protective cultures designated PC1 (Lactobacillus spp.), PC2 (Lactobacillus rhamnosus), and PC3 (Lactobacillus rhamnosus) as biopreservatives in queso fresco (QF) against 9 yeast strains (Candida zeylanoides, Clavispora lusitaniae, Debaryomyces hansenii, Debaryomyces prosopidis, Kluyveromyces marxianus, Meyerozyma guilliermondii, Pichia fermentans, Rhodotorula mucilaginosa, and Torulaspora delbrueckii) and 11 mold strains (Aspergillus cibarius, Aureobasidium pullulans, Penicillium chrysogenum, Penicillium citrinum, Penicillium commune, Penicillium decumbens, Penicillium roqueforti, Mucor genevensis, Mucor racemosus, Phoma dimorpha, and Trichoderma amazonicum). All fungal spoilage strains were previously isolated from dairy processing environments. A positive control (C) with no protective culture was included. Fungal spoilage organisms were inoculated on cheese surfaces at an inoculum level of 20 cfu/g, and cheeses were stored at 6 ± 2°C throughout the study. For yeast enumeration, cheeses were sampled on d 0, 7, 14, and 21 postinoculation. Significant inhibition was detected for each yeast strain by comparing yeast counts for each cheese treated with protective culture against the control cheese using one-way ANOVA with Bonferroni correction performed individually at d 7, 14, and 21 postinoculation. Mold growth was visually observed and imaged weekly through 70 d postinoculation. Whereas PC3 inhibited Cl. lusitaniae, Mey. guilliermondii, and Ph. dimorpha, PC2 inhibited the outgrowth of Cl. lusitaniae, D. hansenii, and Ph. dimorpha. Protective culture 1 had the broadest spectrum of efficacy across yeast and molds, delaying spoilage caused by 4 distinct yeast strains (Cl. lusitaniae, D. hansenii, D. prosopidis, and Mey. guilliermondii), and inhibiting visible growth of 2 mold strains (P. chrysogenum and Ph. dimorpha). Results demonstrated that commercial protective cultures vary in performance, as indicated by the breadth of mold and yeast inhibition at both the genus and species level. This study suggests that manufacturers looking into using protective cultures should investigate their efficacy against specific fungal strains of concern. Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.