Bacterial colonization on biomaterials is a major issue, leading to approximately 20% of implant failures due to infection and biofilm formation. To address this, peptide-based hydrogels incorporating tailored bioactive peptides have emerged as promising candidates for applications in tissue engineering and infection control. Here, we have designed peptide sequences that incorporate i) a self-assembling unit (SaU) and ii) bioactive motifs, including the well-known arginine-glycine-aspartate (RGD) sequence to promote cell adhesion or an antimicrobial peptide derived from lactoferrin (LF) to exhibit antibacterial properties. To aid in the gelation, these peptides were combined with hyaluronic acid (HA), rendering peptide-based hydrogels without the need for additional external assembly triggers, simplifying their application in biomedical contexts. This protocol allowed for a spontaneous formation of a 3D fibrillar network, with structural and physicochemical properties suitable for tissue engineering. The biological evaluation revealed the ability of RGD-based hydrogels to increase the adhesion and spreading of osteoblastic cells compared to controls, while the LF-based hydrogels significantly reduced the viability and attachment of both Gram-positive and Gram-negative strains, clearly affecting bacterial morphology. This report demonstrates the feasibility of this technology to produce hydrogels incorporating distinct biological cues, highlighting their potential as versatile biomaterials to address diverse biomedical challenges.