Multidrug-resistant (MDR) bacteria have posed a major clinical challenge, as many currently available antibiotics have lost their effectiveness. The development of new or improved antimicrobial formulations are required to address this problem. Collected nosocomial multidrug-resistant bacteria were tested for their susceptibility to various aminoglycosides to determine the minimum inhibitory concentrations (MICs). The most potent isolate producing a modifying enzyme inhibitor was identified. The conditions for maximal inhibitory activity were optimized, and the inhibitory protein was subsequently purified and combined with antibiotics to formulate an active preparation against resistant bacteria. The cytotoxicity of the inhibitor was evaluated using a human skin cell line. Streptococcus pneumoniae (B25) exhibited resistance to amikacin, tobramycin, gentamicin, streptomycin, neomycin, kanamycin, and paromomycin, with MICs of 500, 440, 320, 170, 110, 200, and 140 µg/mL, respectively. Similarly, Staphylococcus aureus (B75) was resistant to the same antibiotics, showing MICs of 80, 140, 200, 470, 440, 500, and 410 µg/mL, respectively. Pseudomonas aeruginosa (B80) was the most effective isolate, exhibiting strong inhibitory activity against both S. pneumoniae (B25) and S. aureus (B75). The inhibitory protein was precipitated at 70% ammonium sulfate saturation, purified by ion-exchange and gel-filtration chromatography, and appeared as a single 20 kDa band on SDS-PAGE. Paromomycin (140 or 410 µg/mL) and streptomycin (170 or 470 µg/mL), when combined with a modifying enzyme inhibitor (61.44 µg/mL), showed a significant increase in antibacterial activity against S. pneumoniae (B25) and S. aureus (B75). The inhibitory protein exhibited less than 5% cytotoxicity toward the human skin fibroblast (HSF) cell line, maintaining approximately 99% cell viability.