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This study investigates the tenderizing effects of bromelain extract (BE) obtained from the top phase of an aqueous two-phase system comprising of 18% PEG 6000–17% MgSO₄ in muscle foods. Uniform-sized chunks of beef, chicken, and squid were marinated with different concentrations of BE (0, 3, 7%, and 20%, w/w) for 1 h at room temperature. The physico-chemical and quality characteristics of the marinated samples were determined. Decreases in pH and moisture content were observed in the samples BE treated, but the TCA-soluble peptides content significantly increased (p < 0.05). Water holding capacity and cooking yields were observed in all of the tested samples, especially when the concentration of BE increased. In addition, a reduction of meat firmness and toughness were also observed in all of the samples when compared to the control (p < 0.05). Electrophoretic patterns also revealed extensive proteolysis and a reduction in number and intensity of the protein bands in all of the treated samples. At the microstructural level, tissue fibers were broken, cell membranes were more strongly degraded, the connections between the sarcolemma and the myofibrils were disappeared, and the generation of numerous gaps could be clearly observed when 20% (w/w) BE was added. The results show that the bromelain extract derived from the two-phase extraction of a pineapple peel could be used as an effective meat tenderizer