Inspired by the natural antimicrobial effect of the topographical features of insect wings, this study prepared urchin-like gold nanoparticles (UGNPs) and deposited them on poly(ε-caprolactone) (PCL)/chitosan (P/C) electrospun nanofiber film to strengthen antibacterial activities of this active packaging. Results showed that L-Dopa was a suitable reducing agent to prepare UGNPs, and the spine length of UGNPs increased from 21.23 to 35.83 nm as the molar ratio of L-Dopa:HAuCl4 increased from 1 to 3. As the nanofiber film was immersed in the nanoparticle solution for a longer time, the UGNP content in P/C nanofibers increased. As the spine length of UGNPs and depositing UGNP content increased, the inhibition rate against S. aureus and E. coli. of P/C nanofiber film increased. In addition, P/C nanofiber film deposited with UGNPs also exhibited good thermal stability, hydrophilicity, mechanical strength, and water vapor permeability, exhibiting its potential as an antibacterial active packaging.