Fresh Hispanic-style cheeses, such as Queso Fresco (QF), have often been implicated with outbreaks with Listeria monocytogenes. Nisin has been widely used in the food industry as a preservative due to its ability to inhibit L. monocytogenes; however, nisin has not been successful at limiting the growth of this pathogen in QF. The objective of this study was to evaluate the efficacy of bioengineered nisin derivatives against L. monocytogenes in QF. Nisin derivatives with a single amino acid substitution at residue 30 (I30X) or 32 (V32X) with positively charged amino acids (H, K, R) were produced in Lactococcus lactis MG1363, an engineered strain having the nisin biosynthetic pathway yet lacking nisA. Commercial nisin (nisin A) and nisin derivatives were evaluated to determine their minimum inhibitory concentration (MIC) and residual nisin after 24 h exposure to pH 7 + 22% milk fat at 37°C. The antilisterial activity of nisin A and nisin derivatives was tested in QF (250 µg nisin/g cheese), and untreated cheeses were included as a control. Cheese curds were inoculated with approximately 4 Log cfu/g of L. monocytogenes cocktail of 5 different foodborne outbreak-associated strains, and L. monocytogenes cells were enumerated by spread plating on PALCAM agar supplemented with ceftazidime, across 7 d of storage at 4°C. All experiments were repeated 3 times with samples prepared in duplicate. Evaluated nisin derivatives exhibited a reduced antilisterial activity in vitro (MIC = 25 µg/mL) compared with nisin A (MIC = 3.125 µg/mL). Residual nisin A was not detected after 24 h of exposure to pH 7 + 22% milk fat, on the contrary, all nisin derivatives evaluated showed residual nisin of 40–75%. After 7 d of cold storage, either nisin I30H and V32R resulted in fewer L. monocytogenes cells in QF with viable counts approximately 0.7 Log cfu/g less than control QF. Similar final Listeria counts were observed in QF added with nisin A and control cheeses. In conclusion, our results support that altering residue 30 or 32 of nisin might result in nisin derivatives with enhanced antilisterial activity in QF.