AbstractThis study evaluated the effects of prior knowledge obtained from multi‐objective optimization on the uncertainty quantification of simulated ecologically relevant hydrologic indices (ERHIs) using a hydrological model. Two experiments were formulated considering contrasting conditions in prior knowledge when implementing Bayesian parameter estimation. In the first experiment, we reproduced a common modeling practice in the absence of previous information where uniform priors are considered when calibrating hydrological and error model parameters. In the second experiment, near‐optimal Pareto solutions from multi‐objective calibration were used to build a multivariate prior distribution for estimating hydrological and error model parameters using Bayesian calibration. In both experiments, a likelihood function considering heteroscedasticity and autocorrelation effects was employed. The Unified Non‐dominated Sorting Algorithm III was used for multi‐objective calibration, whereas the multiple‐try differential evolution adaptive metropolis algorithm was used for Markov Chain Monte Carlo sampling. The experiments were tested using a hydrologic model. We selected a subset of ERHIs comprised of 32 central tendency Indicators of Hydrologic Alteration and seven additional indices describing basic streamflow statistical properties. In general, using near‐optimal Pareto parameter distributions as prior knowledge in Bayesian calibration reduced both the bias and variability ranges in ERHIs prediction. Furthermore, there was no significant loss in the reliability of streamflow predictions (from 2% to 6%, p = 0.07) when targeting the selected ERHIs, while improving precision (from 66% to 58%, p < 0.05) and reducing the bias (from 6% to 4%, p < 0.05). Moreover, parametric uncertainty substantially shrank by 72% when linking multi‐objective calibration and Bayesian parameter estimation.