Novel polymer networks composed of biocompatible, hydrophilic poly(2-isopropenyl-2-oxazoline) (PiPOx), poly(ethylene oxide) (PEO), and selected biologically active compounds (cinnamic acid, benzoic acid or eugenol) were developed for potential antimicrobial applications. The applied crosslinking method, based on the addition reaction between oxazoline pendant groups and chosen reagents containing carboxyl functions, is relatively facile, free from by-products, and thus well suited for biomaterial preparation. The one-step synthesis enabled efficient network formation with high gel contents (>90%). The chemical structure of the newly synthesized networks was characterized using Fourier Transform Infrared-attenuated Total Reflection spectroscopy (FTIR-ATR) and 13C Magic-Angle Spinning (MAS) NMR. To evaluate the suitability for biomedical applications, swelling in water and the mechanical properties of the networks were investigated. The antimicrobial efficacy of the prepared hydrogels was tested in neutral medium both by the agar diffusion method and in the liquid culture against Gram-positive and Gram-negative strains: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloaceae. All the tested hydrogels showed an antimicrobial effect in the direct contact zone. Moreover, the eugenol loaded hydrogel expressed a broader bacteriostatic effect inhibiting microorganism growth beyond the contact zone. These form-stable hydrogels with antibacterial properties may be of interest for designing materials dedicated to biomedical applications.