Peritoneal adhesions are a common complication following pelvic and abdominal surgery, which could be reduced using anti-adhesion barriers. However, the shortcomings of existing hyaluronic acid (HA)-based anti-adhesion film, such as the rapid degradation rate and poor operation ability, cannot be ignored. The aim of this study is to develop a dual crosslinked electrospun HA nanofibrous membrane (NFM) with improved properties and prolonged degradation time as an anti-adhesion barrier film for preventing post-surgical peritoneal adhesions. The HA NFMs were ionically crosslinked with FeCl3 (HAF NFMs) and further covalently crosslinked with 1,4-butanediol diglycidyl ether (BDDE) to produce HA-Fe-BDDE (HAFB) NFMs. The membranes were characterized for their physico-chemical properties to confirm the crosslinking mechanisms through scanning electron microscopy with energy dispersive X-ray analysis, infrared spectroscopy, thermal gravimetric analysis, X-ray photoelectron spectroscopy and mechanical properties. In vitro degradation studies confirmed that only the dual crosslinked HAFB membranes demonstrated an appropriate degradation rate, which could maintain the morphology and porous structure after immersion in phosphate buffered saline and cell culture medium for up to 7 days. In vitro studies indicated that HAFB NFMs showed excellent effectiveness in preventing fibroblast penetration and attachment while preserving high biocompatibility without influencing cell proliferation. The peritoneal anti-adhesion efficacy of HAFB NFMs was tested by implanting them in the abdominal cavities of Sprague Dawley (SD) rats. The membrane could exert its barrier effect for a week to significantly reduce the development of peritoneal adhesion compared with HAF NFMs and Seprafilm®. The results show the potential of the novel dual crosslinked HA-based NFMs in prolonged adhesion prevention.