Perchlorate is a widespread environmental contaminant having both anthropogenic and natural sources. Stable isotope ratios of O and Cl in a given sample of perchlorate may be used to distinguish its source(s). Isotopic ratios may also be useful for identifying the extent of biodegradation of perchlorate, which is critical for assessing natural attenuation of this contaminant in groundwater. For this approach to be useful, however, the kinetic isotopic fractionations of O and Cl during perchlorate biodegradation must first be determined as a function of environmental variables such as temperature and bacterial species. A laboratory study was performed in which the O and Cl isotope ratios of perchlorate were monitored as a function of degradation by two separate bacterial strains (Azospira suillum JPLRND and Dechlorospirillum sp. FBR2) at both 10 °C and 22 °C with acetate as the electron donor. Perchlorate was completely reduced by both strains within 280 h at 22 °C and 615 h at 10 °C. Measured values of isotopic fractionation factors were ε¹⁸O = −36.6 to −29.0‰ and ε³⁷Cl = −14.5 to −11.5‰, and these showed no apparent systematic variation with either temperature or bacterial strain. An experiment using ¹⁸O-enriched water (δ¹⁸O = +198‰) gave results indistinguishable from those observed in the isotopically normal water (δ¹⁸O = −8.1‰) used in the other experiments, indicating negligible isotope exchange between perchlorate and water during biodegradation. The fractionation factor ratio ε¹⁸O/ε³⁷Cl was nearly invariant in all experiments at 2.50 ± 0.04. These data indicate that isotope ratio analysis will be useful for documenting perchlorate biodegradation in soils and groundwater. The establishment of a microbial fractionation factor ratio (ε¹⁸O/ε³⁷Cl) also has significant implications for forensic studies.