Two-dimensional nanomaterials (2D NMs) refer to nanomaterials that possess a planar topography with a thickness of one or several atomic layers. Due to their large specific surface areas, atomic thickness, rough edges, and electron confinement in two dimensions, they have emerged as promising antimicrobial agents over antibiotics in combating bacterial infections. However, 2D NMs encounter issues such as low bio-safety, easy aggregation, and limited tissue penetration efficiency. To address these concerns, hydrogels with three-dimensional (3D) networks have been developed to encapsulate 2D NMs, aiming to enhance their biocompatibility, biodegradability, and ability to regulate and remodel the tissue microenvironment at the infected site. This review systematically summarizes the current studies on 2D NM-based antibacterial hydrogels with 3D network structures (named 2N3Hs). Firstly, we introduce the emerging types of 2N3Hs and describe their antibacterial actions. Subsequently, we discuss the applications of 2N3Hs in three biomedical fields, including wound dressing, cancer treatment, and bone regeneration. Finally, we conclude the review with current challenges and future developments for 2N3Hs, highlighting their potential as a promising choice for next-generation biomedical devices, particularly in the field of tissue engineering and regenerative medicine. This review aims to provide a comprehensive and panoramic overview of anti-infective 2N3Hs for various biomedical applications.