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Researchers have extensively studied borate bioactive glass (BBG) for bone regeneration and wound healing applications. In the current study, 13-93B3 (54.6% B₂O₃, 22.1% CaO, 7.9% K₂O, 7.7% MgO, 6.0% Na₂O, and 1.7% P₂O₅) was synthesized using a sol–gel technique and doped with different molar concentrations of Cu (0.01, 0.05, and 0.25 M) into BBG for possible biomedical applications. Then, the antibacterial activity was tested against <i>E. coli</i> and <i>S. aureus</i>. The maximum zone of inhibition against <i>S. aureus</i> was achieved at 100 μg/mL of 0.25 M Cu-doped BBG. At 50 μg/mL of 0.25 molar copper concentration, <i>E. coli</i> showed a significant reduction in colony-forming units. Hydroxyl radical production, influenced by the BBG powder, was most effective against <i>S. aureus</i>, followed by <i>E. coli</i>. Protein leakage studies demonstrated significant leakage after treatment with BBG powder, demonstrating a strong effect on bacterial strains. This shows a change in protein synthesis, which is essential for central metabolism and gene transcription, affecting proteins in the periplasm and inner and outer membranes. Furthermore, carbohydrate leakage studies showed that BBG is effective against all three categories of cellular carbohydrate, namely membrane-bound, transmembrane, and intracellular carbs. This study focuses on the diverse antibacterial processes of Cu-doped BBG, which has emerged as a promising contender for biological applications that require strong antibacterial characteristics.