Textile effluents, although their composition can vary considerably, typically contain high levels of dissolved salts and exhibit wide variations in pH. Ecotoxicological studies regarding the effects of these parameters, however, have been limited owing to the need for sensitive and easy-to-handle bioindicators that require low amounts of sampling, are cost-effective, time-efficient, and ethically endorsed. This kind of study, additionally, demands robust multi-factorial statistical designs that can accurately characterize the individual and combined relationship between variables. In this research, Response Surface Methodology (RSM) was used to calculate the individual and interaction effects of NaCl concentration and pH value of a Simulated Textile Effluent (STE) on the development rate (DR) of the bioindicators: Bacillus subtilis bacteria and Lactuca sativa lettuce. The results demonstrated that the bioindicators were sensitive to both NaCl and pH factors, where the relative sensitivity relationship was B. subtilis > L. sativa. The quadratic equations generated in the experiments indicated that increased concentrations of 50-250 mg L−1 of NaCl caused a perturbance of 1.40%–34.40% on the DR of B. subtilis and 0.50%–12.30% on L. sativa. The pH factor at values of 3–11 caused an alteration of 27.00%–64.78% on the DR of the B. subtilis and 51.37%–37.37% on the L. sativa. These findings suggest that the selected bioindicators could serve as effective tools to assess the ecotoxicological effects of textile effluents on different ecological systems, and the RSM was an excellent tool to consider the ecotoxicological effects of the parameters and to describe the behavior of the results. In conclusion, the NaCl and pH factors may be responsible for disrupting different ecosystems, causing imbalances in their biodiversity and biomass. Before discharge or reuse, it is suggested to remove salts and neutralize pH from textile effluents and, mostly, develop novel, eco-friendlier textile processing techniques.