Subtilisin was immobilized on polycaprolactam and used for food packaging applications to reduce the transference of microorganisms from the packaging material to the packaged food material. The optimized conditions for subtilisin immobilization was as follows: pH, 8; temperature, 4 °C; glutaraldehyde, 0.5%; incubation time, 25 h; and subtilisin concentration, 600 μL. The formation of -CH=N- at 1576 cm -1 in the Fourier transform infrared (FTIR) spectrum confirmed the immobilization. Subtilisin-immobilized polycaprolactam (SIP) exhibited the highest residual activity of 106.67 ± 4.41% and 104.67 ± 0.88% at 40 °C and pH 8 and retained residual activity of 94% at the end of 56 days when compared to 21.33 ± 4.10% in the case of free subtilisin. SIP significantly (p < 0.05) lowered the colony forming units (CFU), dry weight, and protein and carbohydrate contents in bacterial and fungal biofilm. Practical application of the SIP on ham steaks at 4 and 20 °C showed a 2-3 times reduction of Staphylococcus aureus as well as Escherichia coli cells in the range of p < 0.05. © 2011 American Chemical Society.