Biofilms are a self-organized, self-structured community of microorganisms that are enclosed in a self-produced polymeric matrix. Due to the intrinsic complexity of biofilms, their artificial preparation has remained a challenge despite practical usefulness as reservoirs for biocatalysts. Herein, autonomously generated artificial biofilms termed BIOMOSAIC (BioInspired, Oxygen-induced Microbial Organization through Self-Assembly at the air/liquid Interface as a Catalyst) films are reported. Similar to bacterial homing, a large number of cells are spontaneously recruited and immobilized into the film, which is autonomously generated at air/liquid interfaces by simple mixing of phenolic polyamine and tyrosinase-expressing Pseudomonas fluorescens. BIOMOSAIC films are self-regenerated, portable, reusable, and freestanding. They are multifunctional depending on types of gene expressions from the cells, catalyzing various reactions such as l-DOPA production, dephosphorylation, and lipid hydrolysis. Furthermore, BIOMOSAIC film exhibits effective crude oil degradation properties, indicating that it can be a new self-producing and self-sustaining platform for bioremediation.