Paraguayan soup is a traditional and emblematic dish widely consumed in Paraguay. Its valorization and potential for industrial-scale production highlight the need to improve preservation methods. This study aimed to evaluate the effect of nisin on microbial growth of mesophilic aerobes, total coliforms, Escherichia coli, Staphylococcus aureus, and Salmonella spp. at intervals of 7 days, culminating on day 40. Sensory attributes such as texture, appearance, color, and consistency were also assessed, comparing nisin-treated samples (which showed the lowest microbial counts) with a control. Microbiological evaluations followed AOAC specifications, and sensory analysis adhered to the Spanish standard UNE-ISO 6658. Nisin exhibited a biopreservative effect on mesophilic aerobes and Staphylococcus aureus in refrigerated soup dough. Significant differences were found in total coliforms between the treated and control groups, although E. coli counts did not vary significantly. Salmonella spp. were not detected in any samples, so nisin’s inhibitory effect could not be determined in this case.Sensory evaluation revealed a preference for the nisin-treated soup in terms of flavor, color, and aroma—but not in consistency. These findings suggest that nisin has strong potential as a biopreservative in the industrial production of refrigerated Paraguayan soup dough ready for baking, contributing to improved microbial safety and sensory appeal. Moreover, incorporating nisin could significantly enhance the preservation and valorization of Paraguay’s traditional culinary products.