The rates of biodegradation of six model naphthenic acids (NAs) by heterotrophic bacteria were compared. Specifically, the biodegradation of cis- and trans- geometric isomers of 4-methylcyclohexaneacetic acids (4MACH), 4-methylcyclohexanecarboxylic acids (4MCCH) and 3-methylcyclohexanecarboxylic acids (3MCCH) in surface water were monitored by gas chromatography. Biodegradation experiments were conducted using Athabasca River water collected in September 2000. The river water was dosed with two 4MACH concentrations, one 4MCCH concentration and one 3MCCH concentration. Each set of samples was also assessed at different pH levels, one dissolved organic carbon (DOC) content and three different temperatures to determine the effects of these parameters on the rate of substrate biodegradation. Controls that were both filtered and autoclaved displayed insignificant losses indicating that depreciation of 4MACH, 4MCCH and 3MCCH from the dosed samples was due solely to biotic factors. First-order biodegradation kinetics was achieved in all cases. The pH and DOC amendments levels did not appear to significantly affect the rate of the model NAs degradation in comparison to ambient conditions. The average rate constant (K) of the non-amended river water samples at room temperature (19 ± 1 °C) of the trans-4MACH and trans-4MCCH isomers were 2.5 × 10−3 and 2.8 × 10−3 hours−1, while the values of K for the corresponding cis- isomers were 0.7 × 10−3 and 0.6 × 10−3 hours−1, respectively. For all the model NAs investigated, the trans isomers degraded more rapidly than the respective cis isomers. The range of K measured at 10 °C and 30 °C of the trans-isomers were between 0.6 × 10−3 to 0.8 × 10−3 and 2.1 × 10−3 to 7.9 × 10−3 hours−1, respectively, while the corresponding values for the cis- isomers were between 0.2 × 10−3 to 0.5 × 10−3 and 1.2 × 10−3 to 1.8 × 10−3 hours−1, respectively. Intramolecular hydrogen bonding which occurs for only the cis- isomers, is the proposed rationale for the observed differences in biodegradation kinetics of the geometric isomers.