Herein, we report the use of sequential layer-by-layer (LbL) assembly to design nanostructured films made of recombinant bacterial membrane fractions (MF), which overexpress cytochrome P450 (CYP) and cytochrome P450 reductase. The ability to incorporate MF in LbL multilayered films is demonstrated by an in situ quartz crystal microbalance with dissipation monitoring using poly-L-lysine or poly-L-ornithine as a polycation. Results show that MF preserve a remarkable CYP1A2 catalytic property in the adsorbed phase. Moreover, atomic force microscopy images reveal that MF mostly adopt a flattened conformation in the adsorbed phase with an extensive tendency to aggregate within the multilayered films, which is more pronounced when increasing the number of bilayers. Interestingly, this behavior seems to enhance the ability of embedded MF to remain active after repeated uses. The proposed strategy constitutes a practical alternative for the immobilization of active CYP enzymes. Besides their fundamental interest, MF-based multilayers are useful nano-objects for the creation of new biomimetic reactors for the assessment of xenobiotic metabolism.