Routine monitoring of the quality status of water bodies demands the best cost-benefit relation and sample-size reduction is therefore welcomed. However, great caution is needed because such reduction affects the accuracy and variation of the results. In the present study we tested the influence of sample size (number of replicate samples) on reference condition values and within-sample ecological quality ratio (EQR) variability of six commonly used ecological indices (taxa richness, Shannon–Wiener diversity, AMBI, Medocc index, Bentix and M-AMBI). Analysis of soft-bottom benthic invertebrate data from Slovenian coastal waters showed that sample size influenced the reference condition values of richness/diversity indices (taxa richness and Shannon–Wiener diversity) but not of the sensitivity/tolerance indices (AMBI, Medocc index, Bentix). Increased sample size decreased the within-sample EQR variability and, concomitantly, increased the accuracy of site ecological status classification for all indices. The size of EQR variability differed depending on the index used. EQR variability of M-AMBI, an index composed of metrics with different within-sample EQR variability, was statistically the same as that of the metric with the lowest within-sample EQR variability. Whether this is a common principle for multimetric indices remains to be confirmed. Based on these results, the use of at least three replicates is suggested to obtain reliable measures of reference condition and EQR for the assessment of ecological status. This level of replication is particularly necessary in areas with high diversity and environmental patchiness, and when richness/diversity measures and indices that include these measures are used.