The development of advanced probiotic delivery systems, which preserve bacteria from degradation of the gastrointestinal tract and achieve a targeted release mediated by pH-independent swelling, is of great interest to improve the efficient delivery of probiotic bacteria to the target tissue. Gram-positive and Gram-negative bacteria models (Lactobacillus acidophilus (Moro) Hansen and Mocquot (ATCC® 4356™) and Escherichia coli S17, respectively) have been successfully encapsulated for the first time in pH-independent microparticulate polymethacrylates (i.e., Eudraguard biotic) used for the targeted delivery of nutraceuticals to the colon. These bacteria have also been encapsulated within the mucoadhesive polymethacrylate Eudragit RS 100 widely used as targeted release formulation for active pharmaceutical ingredients. The enteric microparticles remained unaltered under simulated gastric conditions and released the contained viable microbial cargo under simulated intestinal conditions. Buoyancies of 90.2% and 57.3% for Eudragit and Eudraguard microparticles, respectively, and long-term stability (5 months) for the encapsulated microorganisms were found. Cytotoxicity of the microparticles formulated with both polymers was evaluated (0.5–20 mg/mL) on Caco-2 cells, showing high cytocompatibility. These results underline the suitability of the synthesized materials for the successful delivery of probiotic formulations to the target organ, highlighting for the first time the potential use of Eudraguard biotic as an effective enteric coating for the targeted delivery of probiotics.