Diatoms are microscopic, unicellular algae that represent dominant photosynthetic organisms in the world’s oceans sustaining marine food web. In contrast to the chemical ecology of planktonic diatoms that is well documented, few studies have reported the effects of benthic diatoms, since they are more difficult to sample, quantify and cultivate than planktonic species. Herein, for the first time we investigated the effects of feeding on two bloom-forming benthic diatoms isolated from the leaves of the seagrass Posidonia oceanica on the sea urchin Paracentrotus lividus. The two benthic diatom species tested (Nanofrustulum shiloi and Cylindrotheca closterium) were identified using Scanning Electron Microscopy and molecular analysis of the 18S rRNA gene. Adult P. lividus were fed one month on these two benthic diatoms and on the green alga Ulva rigida, used as feeding control. We collected eggs and sperm from adult P. lividus and after fertilization we measured fertilization success, the percentage of first mitotic cleavage and embryonic development until the pluteus stage. 1H-NMR metabolomic analysis of aqueous and lipophilic extracts were performed on gonad tissues from adults fed for one month with the two benthic diatoms compared with those fed with U. rigida. We then generated de novo transcriptome assembly and annotation of P. lividus embryos at the pluteus stage (deriving from adults fed for one month with the two benthic diatoms) to identify differentially expressed genes. We also followed, using real-time quantitative PCR, fifty genes belonging to different functional classes to identify molecular targets and to validate RNA-seq results. Our results demonstrate the toxigenic effects of benthic diatoms upon grazing activity of the sea urchin P. lividus. Interestingly, preliminary results indicate that the toxins are not PUAs or canonical oxlipins that have already been identified in planktonic diatoms as inducing reproductive failure, opening new perspectives to find new oxylipins or other compounds.