By understanding the nutritional requirements for pathogenic bacteria, researchers can effectively cultivate and study them as well as explore potential antimicrobial agents that may combat them. A new bacterial culture medium was prepared using a cost-effective and available type of fungus, Terfezia spp. At the same time and using the green method, titanium oxide nanoparticles (TiO2 NPs) were mycosynthesized using the same fungus. In a novel manner, the TiO2 NPs were loaded into polyvinyl alcohol/sodium alginate (PVA/SA/TiO2 NPs) aerogel beads, which were prepared using the sol-gel method and freeze-drying cycle. These nanocomposite aerogel beads were characterized using several techniques such as UV-vis, FTIR, SEM, and XRD. Bacterial growth analysis showed successful growth of all pathogenic bacterial samples on the Terfezia spp. culture medium. The UV-vis analysis for PVA/SA/TiO2 NPs exhibiting a characteristic peak within 260–290 nm. FTIR characterization demonstrated the successful mycosynthesis of TiO2 NPs using Terfezia spp. and interaction between the TiO2 NPs and polymer. SEM revealed the TiO2 NPs had spherical morphology with an average size around 38 nm while the aerogel bead surface showed a uniform nanoporous structure. XRD analysis indicated the TiO2 NPs was incorporated into the PVA/SA matrix. The mycosynthesized TiO2 NPs exhibited broad-spectrum antibacterial activity against pathogenic bacterial strains, with zones of inhibition ranging between 24–34 mm, while the antimicrobial activity of the PVA/SA/TiO2 NPs aerogel beads ranged between 12–20 mm, as indicated by the well diffusion assay. The minimum inhibitory concentration (MIC) of the TiO2 NPs was found to range between 16–64 μg/mL, while the minimum bactericidal concentration (MBC) values were determined to be between 8–32 μg/mL for the tested pathogenic bacterial strains. Growth kinetics testing showed that the PVA/SA/TiO2 NPs aerogel beads exhibited complete inhibition against all pathogenic bacterial strains, in contrast to the pure PVA/SA aerogel beads. The antibacterial performance was directly related to the loading of the TiO2 NPs in the polymer matrix. Finally, the enhanced antibacterial activity of these nanocomposite aerogel beads suggests they could be utilized for antibacterial materials and biomedical applications.