Abstract We developed a zooplankton-based water-quality evaluating method using indices of alpha diversity. Two key objectives were set: (i) the comparison of two—different quality—samples from different areas, and the verification of their differentiation, based on mesozooplankton biodiversity indices; and (ii) the development of a methodology, which was able to assess the quality of new marine water samples. Our analysis was based on a 24-year-long in situ dataset (1987–2010) of 139 samples in which 86 mesozooplankton taxa were identified. High-diversity and high evenness values were reported in the case of the “good” status sample, while low diversity, low evenness and high dominance values occurred at the lower quality one. A linear discriminant analysis (LDA) was conducted that discriminated the tested samples at 100%. This LDA was then used to evaluate samples of unknown quality. Finally, 90% of them were classified with a probability of correct classification (posterior probability) >95%. The present study proves that mesozooplankton diversity indices can discriminate different levels of anthropogenic impacts. In this sense, it can be used as a reliable indicator for environmental assessment in the pelagic habitats of the Mediterranean Sea.