Chitosan has been successfully used as a biomaterial with several purposes in many species. In this study, chitosan membranes were produced with six different types of materials, and their behavior were evaluated upon implantation in the subcutaneous tissue of the flank of twelve healthy horses. We assessed chitosan membranes obtained from commercial chitosan, impregnated or not with silver nanoparticles, sterilized with ethylene oxide (CCEO, n=3; CCSNEO, n=3) or by ultraviolet radiation (CCUR, n=3; CCSNUR, n=3), and chitosan membranes obtained from squid gladius, sterilized with ethylene oxide (SCEO, n=6) or by ultraviolet radiation (SCUR, n=6). The same animals were randomly used in two experimental groups, with a minimum interval of 60 days between procedures, respecting the fact of only one flank side, left or right, be under evaluation by experimental period. After preparation of the membranes and implantation in the flank subcutaneous tissue of the horses, macroscopic and ultrasonographic evaluations of the implant regions were performed, as well as physical examination, blood count and fibrinogen measurement. No clinical or laboratory abnormalities were observed. All animals that received commercial chitosan membranes, regardless of the preparation technique, showed rejection to the biomaterials, considering that 100% of the surgical wounds presented dehiscence of suture and expulsion of the implants. The animals that received squid gladius chitosan membranes showed success in the treatment, with healing by primary intention of the surgical wound. We conclude that squid gladius chitosan membranes are biocompatible and biodegradable when implanted in the subcutaneous tissue of the flank of healthy horses.