The photoantibacterial properties of titania nanoparticles (TiO2NPs) are attracting much interest, but the separation of their suspension limits their application. In this study, the encapsulation of commercial TiO2NPs within self-assembling tripeptide hydrogels to form hgel-TiO2NP composites with significant photoantibacterial properties is reported. The Fmoc-Phe3 hydrogelator was synthesized via an enzymatic method. The resulting composite was characterized with DLS, ζ-potential, SAXS, FESEM-EDS and rheological measurements. Two different concentrations of TiO2NPs were used. The results showed that, by increasing the TiO2NP quantity from 5 to 10 mg, the value of the elastic modulus doubled, while the swelling ratio decreased from 63.6 to 45.5%. The antimicrobial efficacy of hgel-TiO2NPs was tested against a laboratory Staphylococcus aureus (S. aureus) strain and two methicillin-resistant S. aureus (MRSA) clinical isolates. Results highlighted a concentration-dependent superior antibacterial activity of hgel-TiO2NPs over TiO2NPs in the dark and after UV photoactivation. Notably, UV light exposure substantially increased the biocidal action of hgel-TiO2NPs compared to TiO2NPs. Surprisingly, in the absence of UV light, both composites significantly increased S. aureus growth relative to control groups. These findings support the role of hgel-TiO2NPs as promising biocidal agents in clinical and sanitation contexts. However, they also signal concerns about TiO2NP exposure influencing S. aureus virulence.