Low availability of oxygen can lead to stalled wound healing processes and chronic wounds. To address local hypoxia and to better understand direct cellular benefits, a perfluorocarbon (PFC) conjugated chitosan (MACF) hydrogel that delivers oxygen was created and applied for the first time to in vitro cultures of human dermal fibroblasts and human epidermal keratinocytes under both normoxic (21% O2) and hypoxic (1% O2) environments. Results revealed that local application of MACF provided 233.8 ± 9.9 mmHg oxygen partial pressure after 2 h then maintained equilibrium oxygen levels that were approximately 17 mmHg partial pressure greater than untreated controls. Cell culture experiments showed that MACF oxygenating gels improved cellular functions involved in wound healing such as cell metabolism, total DNA synthesis and cell migration under hypoxia in both fibroblasts and keratinocytes. Adenosine triphosphate (ATP) quantification also revealed that MACF treatments improved cellular ATP levels significantly over controls under both normoxia and hypoxia (p<0.005). In total, these studies provide new data to indicate that supplying local oxygen via MACF hydrogels under hypoxic environments improves key wound healing cellular functions.