ABSTRACT Media formulation ingredients are often identified as potential sources of contamination by microorganisms in cellular agriculture. Therefore, this study aimed to evaluate the thermal tolerance of foodborne pathogens cultured in Leibovitz's L‐15 Medium with varying Fetal Bovine Serum concentrations (0%, 2%, and 10%) at 36°C, subjecting the media formulations to thermal treatments at 55°C, 60°C, 65°C, 70°C, and 75°C. The highest thermal inactivation gradients for bacteria were observed in media containing 10% FBS, suggesting that the presence of 10% FBS plays a significant role in increasing bacterial susceptibility to thermal treatment. Escherichia coli cultured in 10% FBS and subjected to 55°C exhibited the highest D ‐value compared to those cultured in 0% and 2% FBS. However, Salmonella cultured in 10% FBS showed opposite trends with the lowest D ‐value. Among the Gram‐positive and Gram‐negative bacteria evaluated, Escherichia coli exhibited the highest z ‐value across all tested media, signifying its exceptional thermal tolerance compared to the other bacteria. Gram‐negative bacteria ( E. coli and Salmonella ) had decreasing z ‐values with higher FBS, while Gram‐positive bacteria ( Listeria and Staphylococcus ) showed opposite trends. This study establishes that bacterial response to serum concentrations significantly impacts thermal treatments in cell culture‐based food processing and storage. The impact of thermally treated samples on cell performance showed no significant difference in cell metabolic activities between control (unpasteurized media) and media pasteurized at 55°C for 75.24 min. However, increasing the temperature significantly reduced cell metabolic activities, with the lowest metabolic activities observed in cells grown in media heated to 75°C for 0.54 min.