Pluronic F-127 (PF-127) hydrogel is a versatile biomaterial with promising applications in drug delivery, tissue engineering, and regenerative medicine. PF-127 has antiadhesive activity that prevents bacterial adhesion by creating a hydrated layer on the bacterial surface. This property makes PF-127 suitable for preventing implant-associated infections. In this study, we aimed to evaluate the antibacterial properties of PF-127 using field isolates of Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria) and compare them with different antibiotic standards. The antimicrobial potential was assessed using disk diffusion assays with four standard concentrations (20%, 25%, 30%, and 40%). The test microorganisms were inoculated on agar plates, and sterile filter paper disks infused with PF-127 hydrogels were placed alongside standard antibiotic disks. After incubation, the inhibition zones were measured to determine antimicrobial activity. Our results showed that PF-127 lacked intrinsic antimicrobial activity against S. aureus and E. coli at the tested concentrations. In conclusion, PF-127 hydrogel is a promising neutral carrier hydrogel system for loading antibiotics and antimicrobial compounds. Its unique properties, such as biocompatibility and thermo-responsive behaviour, combined with its antiadhesive activity, make it an ideal candidate for various biomedical applications.