We previously reported that d-tryptophan exhibits adverse effects on bacterial physiology under osmotic stress. However, the mechanism by which d-tryptophan acts as an inhibitor and/or incompatible solute for bacterial growth has not yet been investigated in detail. In this study, we aimed to determine how osmotic pressure and temperature affect the antimicrobial effect of d-tryptophan. Even at the same level of osmotic pressure, d-tryptophan in conjunction with sodium chloride (NaCl) had a stronger inhibitory effect on the growth of Escherichia coli than that obtained by incubation with potassium chloride (KCl) and sucrose. Because d-tryptophan with NaCl showed the strongest inhibitory effect, we determined the optimum concentration combination of d-tryptophan and NaCl. The growth inhibition boundary conditions as a function of d-tryptophan and NaCl concentrations were determined by a logistic regression model. We found that the minimum level of NaCl for E. coli growth inhibition was 2.5% (w/v) together with 40 mM d-tryptophan. Moreover, the higher the NaCl concentration, the lower the concentration of d-tryptophan that was needed to inhibit bacterial growth. The logistic regression model that we developed enabled us to predict the concentrations required to inhibit bacterial growth. Furthermore, we examined the effect of incubation temperatures ranging from 15 to 46°C on the antimicrobial effect of d-tryptophan. The higher the reaction temperature, the more rapid the decrease of viable E. coli that was observed. This trend is likely attributable to activation of physiological metabolism under the optimum growth temperature. Together, our findings should make a significant contribution to the development of a novel bacterial growth control strategy using d-tryptophan.