Ecologically relevant streamflow characteristics (SFCs) of ungauged catchments are often estimated from simulated runoff of hydrologic models that were originally calibrated on gauged catchments. However, SFC estimates of the gauged donor catchments and subsequently the ungauged catchments can be substantially uncertain when models are calibrated using traditional approaches based on optimization of statistical performance metrics (e.g., Nash–Sutcliffe model efficiency). An improved calibration strategy for gauged catchments is therefore crucial to help reduce the uncertainties of estimated SFCs for ungauged catchments. The aim of this study was to improve SFC estimates from modeled runoff time series in gauged catchments by explicitly including one or several SFCs in the calibration process. Different types of objective functions were defined consisting of the Nash–Sutcliffe model efficiency, single SFCs, or combinations thereof. We calibrated a bucket-type runoff model (HBV – Hydrologiska Byråns Vattenavdelning – model) for 25 catchments in the Tennessee River basin and evaluated the proposed calibration approach on 13 ecologically relevant SFCs representing major flow regime components and different flow conditions. While the model generally tended to underestimate the tested SFCs related to mean and high-flow conditions, SFCs related to low flow were generally overestimated. The highest estimation accuracies were achieved by a SFC-specific model calibration. Estimates of SFCs not included in the calibration process were of similar quality when comparing a multi-SFC calibration approach to a traditional model efficiency calibration. For practical applications, this implies that SFCs should preferably be estimated from targeted runoff model calibration, and modeled estimates need to be carefully interpreted.