While the body has an incredible ability to heal, host and external factors may overwhelm its innate regenerative capacity. In these instances, a tissue defect may occur. Tissue defects (regions of either necrotic tissue or void space) are highly susceptible to microbial invasion. Given their proximity to the native micro- and mycobiome, craniofacial and cutaneous defects are at particularly high risk for chronic contamination and infection. The combination of tissue loss and infection creates a negative feedback loop: 1) lack of vascularized healthy tissue leads to a locally immunocompromised area; 2) pathogens are able to colonize the tissue defect and eventually invade the margins of healthy tissue; 3) the resulting pathogenic attack and inflammatory response results in tissue injury and necrosis at the defect border; and 4) the tissue defect expands. Two possible mechanisms to break this cycle are to restore vascularized tissue to the defect site or clear the infection to restore the body’s ability to regenerate tissue. In this dissertation, we seek to develop technologies to treat large tissue defects susceptible to infection through biomaterials-based strategies. First, we investigated and optimized the in vivo bioreactor platform for generating autologous free tissue flaps for mandibular reconstruction. As the engineered tissues grown in these bioreactors are vascularized, their use in mandibular repair restores circulation to the defect site. In a large animal model of disease, we demonstrated that these bioreactors did not require harvested donor tissue, exogenous stem cells, or growth factors in order to generate bony flaps suitable for reconstruction. When transferred to a large mandibular defect in a physiologically-relevant ovine model, these engineered tissues were capable of integrating with the native host tissue for functional craniofacial repair. In the second aim of this work, space maintenance was explored to facilitate the repair of large tissue defects by stimulating the growth of a healthy soft tissue envelope around the defect space as well as functioning as a depot for local delivery of antimicrobial agents to prevent and/or treat infection of the vulnerable large tissue defect. Porous space maintainers were fabricated per good manufacturing practice, subjected to electron beam irradiation, and evaluated for suitability of subsequent mechanical properties. These porous space maintainer devices were then implanted in a superior marginal defect adjacent to the oral mucosa in the mandibular diastema of an ovine model of disease. After nine weeks, space maintainers were removed and the defect was reconstructed with tissue-engineered vascularized flaps generated in 3D-printed bioreactors. Even in a challenging defect environment with proximity to the oral flora and under mechanical load, the space maintainers were able to prevent collapse of tissue into the defect site and maintain a healthy soft tissue envelope for repair. Infection was associated with the single failed case during the use of this strategy. Given the high risk of infection in the setting of large craniofacial defects, econazole-eluting porous space maintainers were developed for the local delivery of an antimicrobial therapeutic during space maintenance. Compared to traditional solid space maintainers, porous space maintainers were able to better inhibit the in vitro growth of common fungal and bacterial pathogens and may be of value in the treatment of tissue defects with infection. Finally, the last aim of this thesis specifically examined novel antifungal approaches for the treatment of infected tissue defects. As there is currently a dearth of animal models of fungal infection relevant to tissue engineering, a murine model with a large cutaneous defect infected with Aspergillus fumigatus was established. A new class of diol-based aliphatic polyesters was synthesized and characterized as polymers whose degradation products have inherent antifungal properties. These diol-based polymers were then fabricated into microparticles and loaded with traditional antifungal therapeutics. After demonstrating extended release of therapeutics in vitro, the microparticles were used to locally treat fungal infection in a large cutaneous defect in an immunocompromised murine model of disease. In sum, this body of work explores biomaterials-based approaches to treat large infected defects, through restoration of tissue and/or by mitigating infection. From leveraging the body’s own innate healing capacity to create vascularized tissue suitable for defect reconstruction (in vivo bioreactors), to utilizing traditional biomaterials in innovative ways (antifungal-eluting bone cement-based space maintainers), to developing new biomaterials with specific antimicrobial applications in mind, we have created a number of strategies to aid in the regeneration of large tissue defects.