Three cheese samples containing L. casei formulated with milk fat (C1), shortening oil (SH1), and cocoa butter substitute oil (SU1) and other three samples containing L. acidophilus formulated with milk fat (C2), shortening oil (SH2), and cocoa butter substitute oil (SU2) were prepared. Cheese samples were evaluated for their chemical composition, pH, and viable counts of L. casei and L. acidophilus during storage for 30 days before and after in vitro digestion. pH values gradually decreased over time (milk fat-based cheese samples had the highest pH values). Results revealed that the viability of Lactobacillus spp. was strain-dependent (L. casei was of higher viability than L. acidophilus). In the undigested samples, the viability of L. casei and L. acidophilus significantly decreased over time, keeping higher viable counts (˃10⁶ CFU/g). In vitro digestion strongly declined Lactobacillus viability and L. acidophilus was the most inhibited strain. Results demonstrated a great influence of fat type on Lactobacillus viability where the highest viable numbers were observed in samples containing milk fat (C1, and C2). Moreover, the inhibition rate (%) was strongly influenced by in vitro digestion, fat type, and Lactobacillus strain used. Accordingly, the higher inhibition rate was recorded for L. acidophilus in digested cheese containing shortening and substitute oils (41.47, and 34.04%, respectively). Thus, the results of the current study indicated that milk fat is the most suitable fat type in protecting probiotic viability in cheese.