PEG-based hydrogels possess tissue-like mechanical elasticity, solute permeability, cytocompatibility and biocompatibility. In this work, PEG-based hydrogels were prepared via nucleophilic thiol-yne addition between a 4-arm PEG functionalized with thiols (PEG10k-4-SH) and an electron-deficient alkyne (PEG10k-4-PP). The as-fabricated hydrogels still possess residual functionalities, enabling a second nucleophilic thiol-yne addition on the gel matrix. A thiol-containing fluorescent dye was conjugated with the electron-deficient alkyne appended hydrogels. A thiol-containing antimicrobial peptide (AMP-SH) was also embedded into the gel matrix via nucleophilic thiol-yne addition. The inhibition of bacterial growth in suspensions and contaminated substrate surfaces by the AMP-embedded hydrogels was studied. The cytotoxicity of unmodified and AMP-embedded PEG-based hydrogels against 3T3 fibroblasts was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability assay.