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The emergence of new antibiotic‐resistant bacterial strains means it is increasingly important to find alternatives to traditional antibiotics, such as bacteriolytic enzymes. The bacteriolytic enzyme lysozyme is widely used in medicine as an antimicrobial agent, and covalent immobilization of lysozyme can expand its range of possible applications. However, information on the effect of such immobilized preparations on whole bacterial cells is quite limited. Here, we demonstrate the differential effects of glycine and charged (basic and acidic) amino acids on the enzymatic lysis of Gram‐positive and Gram‐negative bacteria by soluble and immobilized lysozyme. Glycine and basic amino acids (histidine, lysine, and arginine) significantly increase the rate of lysis of Gram‐negative Escherichia coli cells in the presence of soluble lysozyme, but they do not substantially affect the rate of enzymatic lysis of Gram‐positive Micrococcus luteus . Glutamate and aspartate significantly enhance enzymatic lysis of both E. coli and M. luteus . When using immobilized lysozyme, the effects of amino acids on the rate of cell lysis are significantly reduced. For immobilized lysozyme, the presence of an external diffusion mode on cell lysis kinetics at bacterial concentrations below 4 × 10 8 colony‐forming units·mL −1 was shown. The broadening of the pH optimum of lysozyme activity after immobilization has been demonstrated for both Gram‐positive and Gram‐negative bacteria. The Michaelis constant ( K m ) values of immobilized lysozyme were increased by 1.5‐fold for E. coli cell lysis and 4.6‐fold for M. luteus cell lysis compared to soluble enzyme. A greater understanding of the effect of amino acids on the activity of native and immobilized lysozyme is important for both the development of new materials for medical purposes and elucidating the interaction of lysozyme with bacterial cells. Of particular interest is our finding that lysozyme activity against Gram‐negative bacteria is enhanced in the presence of glycine and charged amino acids over a wide range of concentrations.