Abstract In this study, nanofibers composed of Opuntia cochenillifera nopal mucilage (N) extract combined with chitosan (CH) and pullulan (PL) (N/CH/PL) were produced via Forcespinning®. The developed nonwoven composite membranes are composed of long, continuous and homogeneous fibers with average fiber diameter varying between 251 ± 77 nm and 406 ± 127 nm depending on the concentration of N. After crosslinking, the developed membranes were highly stable in water. The water absorption capacity of the N/CH/PL composite nanofiber membranes was shown to be 65% higher compared to CH/PL nanofiber membranes. Nopal dip‐coated membranes show inhibition of Gram‐negative Escherichia coli , indicating antibacterial properties. These findings suggest that the incorporation of naturally derived nopal extract into nanofiber systems could provide a natural alternative for dressings used in wound healing applications. © 2020 Society of Chemical Industry