This study aimed to investigate the effect of using mesophilic culture consisting of Lactococcus lactis sub. lactis and Lactococcus lactis sub. cremoris, and thermophilic culture consisting of Lactobacillus delbrueckii sub. bulgaricus and Streptococcus thermophilus, either single or in combination, on the quality and safety of Sudanese Muddaffara cheese during a ripening period. The cheese is made with mesophilic culture (T1), thermophilic culture (T2), and mixed mesophilic and thermophilic culture (T3). Cheese was sampled at day 0, 30, 60, and 90 of ripening periods and evaluated for physiochemical, microbiological, texture and sensory characteristics. The results indicated that the physicochemical characteristics of Muddaffara cheese vary depending on the starter culture used. The acidity of cheese with mesophilic cultures ranged from 1.03-1.33%, while with thermophilic culture ranged from 1.50 -1.71%.. The total viable count (TVC) gradually increased during storage in all treatments, with the highest counts observed for the mixed mesophilic and thermophilic culture, followed by the thermophilic culture and mesophilic culture. Cheese produced with mesophilic culture showed a hardness value of 9.32 N, which was decreased to 8.80 and 7.89 N in the cheese produced with thermophilic and mixed culture, respectively. Muddaffara cheese was highly acceptable when produced with mixed lactic culture followed by thermophilic culture and lastly with mesophilic lactic culture. Overall, the results of this study suggest that the use of mixed mesophilic and thermophilic cultures could be a promising approach to improve the quality and safety of Sudanese Muddaffara cheese.