Fresh Hispanic-style cheeses (FHSC), such as Queso Fresco (QF), have been implicated in several outbreaks linked to Listeria monocytogenes, and effective biocontrol measures are needed to improve FHSC safety. The objectives of this study were to investigate the potential synergy between endolysin PlyP100 and nisin against L. monocytogenes in QF, and to examine pathogen resistance development after exposure to PlyP100 and nisin in QF. His-tagged PlyP100 was overexpressed in Escherichia coli and subsequently purified. PlyP100 and nisin were added to miniature QF at the following concentrations: 2.5 or 10 U/g PlyP100 with or without 250 µg/g nisin. Antilisterial activity of antimicrobial combinations were tested by inoculating cheese curds with approximately 4 Log cfu/g of L. monocytogenes cocktail, and survival of the pathogen was measured across 28 d of storage at 4°C. All experiments were repeated 3 times with samples prepared in duplicate. By the end of QF storage, 3 random L. monocytogenes isolates per cheese treatment, per independent experiment, were cultured and their sensitivity to PlyP100 and nisin was tested. PlyP100 reduced viable counts of L. monocytogenes in QF by up to approximately 1 Log cfu/g, and no regrowth was observed during 28 d storage. Nisin alone was ineffective to control the pathogen in QF, leading to subsequent regrowth. All treatments combining nisin and PlyP100 in QF achieved reduction of L. monocytogenes below the detection limit of plating. Additionally, in half of the QF samples with nisin + PlyP100, the pathogen was not recovered after enrichment. No difference in sensitivity to nisin or PlyP100 was observed in 36 random L. monocytogenes isolates from QF samples, regardless of whether antimicrobials were added to QF. In conclusion, our results support the use of phage endolysin combined with nisin as a more efficient Listeria control measure in QF.