1. To mitigate the ongoing threats to coastal ecosystems, and the biodiversity erosion they are causing, marine-protected areas (MPAs) have emerged as powerful and widespread conservation tools. Strictly no-take MPAs, also called marine reserves, undeniably promote fish biomass and density, but it remains unclear how biodiversity responds to protection. Identifying which facets of biodiversity respond to protection is critical for the management of MPAs and the development of relevant conservation strategies towards the achievement of biodiversity targets.2. We collected 99 environmental DNA (eDNA) samples inside and outside nine marine reserves in the Mediterranean Sea to assess the effect of protection on 11 biodiversity indicators based on fish traits, phylogeny and vulnerability to fishing. We controlled for the effect of environmental heterogeneity (habitat, bathymetry, productivity, temperature and accessibility) using a principal component analysis, and for spatial autocorrelation due to potential unmeasured factors.3. We found a positive and significant effect of protection on only 3 out of 11 indicators: functional and phylogenic diversity but also the ratio between demersopelagic and benthic species richness. Rather, total fish richness responded significantly and negatively to protection. We did not detect any significant effect of protection on threatened and elasmobranch species richness, probably due to their large home range compared to the size of Mediterranean marine reserves.