Physically crosslinked poly(vinyl alcohol)-hyaluronic acid (PVA-HA) hydrogel membranes composed of different amounts of HA were prepared by freeze-thawing (F-T) method. F-T cycle was repeated for three consecutive cycles. HA was chosen and routinely utilized in the local treatment of chronic wounds, because of its advantages as, HA is endogenous and biodegradable polymer. Physicochemical properties of PVA-HA membranes such as, gel fraction (GF), swelling, mechanical properties, hydrolytic degradation and in vitro bio-evaluation tests were investigated. Results revealed that introducing HA into PVA structure affected significantly the physicochemical properties of membranes than the pristine PVA, because of its crosslinking interaction with PVA. With the increase of HA content in PVA hydrogel membranes, GF and mechanical stability of PVA-HA membranes decreased. However, the swelling behavior, mechanical flexibility, protein adsorption and hydrolytic degradation of PVA membrane increased. The HA content < 20% in PVA hydrogels showed high cell viability (%) and no toxicity was observed using microculture tetrazolium assay (MTT-assay). However, less cell viability was determined with high HA incorporation. PVA-HA-ampicillin free showed antimicrobial activity against Candida albicans as a result of HA presence. Thus, ampicillin-loaded wound dressing with PVA-HA membranes could be used as promising materials with easy forming and biologically evaluated for wound care