Carvacrol is a natural essential oil with a monoterpenoid structure and draws attention due to its antimicrobial and antioxidant capacity. However, high volatility and hydrophobicity limit its use in food packaging systems. This hindrance can be overcome by the complexation with cyclodextrins. In this study, the inclusion complexes (IC) of gamma-cyclodextrin (γCD) and carvacrol were integrated into gelatin/pullulan nanofibers. The control sample of carvacrol loaded gelatin/pullulan nanofibers were generated, as well. Both nanofibers indicated a free-standing and flexible character with defect-free morphology. The carvacrol-γCD-IC crystals were obviously detected within the gelatin/pullulan nanofiber structure differently from carvacrol loaded one. The inclusion complexation of γCD with carvacrol decreased the loss of this essential oil during electrospinning significantly (p < 0.05). Carvacrol retention was determined as 67.84% and 57.63% after two months of storage at room temperature for the carvacrol-γCD-IC and carvacrol loaded gelatin/pullulan nanofibers, respectively. Here, inclusion complexation played a key role in enhancing thermal stability and antibacterial performance of carvacrol loaded in the gelatin/pullulan nanofibers. The promising antioxidant property of nanofibers was revealed in food packaging applications by the accelerated shelf-life test at 40 °C. Oxidation of fish oil samples was retarded by carvacrol-γCD-IC loaded nanofibers. This study provided an understanding of the potential of carvacrol in active food packaging and how the inclusion complex with CD affected the physicochemical properties of this bioactive compound.