The use of activated sludge to assess the potential toxicity and environmental impact of chemicals and wastewaters suffers from several drawbacks related to the heterogeneity, absence of standardization, and health risk associated with this mixed-sewage population. To search for reliable testing inoculum alternatives, the performance of two commercial inocula (BI-CHEM and BIOLEN M112), a garden-soil inoculum and a pure culture of Pseudomonas sp., was compared with an activated sludge inoculum (AS) in the inhibition respiration test ISO 8192-1986 (E). The respiration rates of microbial inocula were assayed for the reference compound 3,5-dichlorophenol. The acute toxicity values (IC(50)) ranged from 6.7 mg/L (Pseudomonas sp.) to 22.7 mg/L (garden soil), overlapping the expected values for activated sludge microorganisms despite the bacterial diversity. The assayed microbial inocula also showed higher reproducibility than AS and an overall similarity of catabolic profiles obtained with Biolog EcoPlates was observed between AS and some mixed inocula. These results point to the potential ability of such inocula as surrogate cultures in relevant activated sludge-based bioassays. New, well-defined, standardized, and safe tools will then be available for monitoring the ecological impact of hazardous substances and effluents, thus providing environmental protection.