As a novel approach in skin tissue engineering, gold nanoparticles (AuNPs) were synthesized and incorporated at different concentrations into collagen/PEO nanofibrous matrices in this study. The group containing 14.27 ppm AuNPs (CM-Au) had the best nanofibrous morphology. CM-Au was cross-linked with glutaraldehyde vapor (CM-AuX). All groups were disrupted in collagenase in 2 h, but cross-linked groups and Matriderm1397904493 resisted hydrolytic degradation for 7 and 14 days, respectively. Due to its small pores and dense structure, lower water swelling results (7.26 ± 2.62 g/g) were obtained for CM-AuX than Matriderm (17.51 ± 1.97 g/g). CM-Au and Matriderm had statistically similar tensile strength and elastic modulus, but elongation at break of CM-Au (over 100%) was significantly better than that of Matriderm. After cross-linking, tensile strength and elastic modulus of collagen matrix was further improved. AuNPs (37 and 42 nm) seemed to be nontoxic on 3T3 fibroblasts and keratinocytes for different time periods. CM-AuX scaffold extracts were also nontoxic for 3T3 fibroblasts and keratinocytes. The L929 cell attachment and proliferation on CM-AuX were comparable with Matriderm, indicating good in vitro biocompatibility. As a whole, collagen matrices incorporated with AuNPs are potential biomaterial candidates for skin tissue engineering.