<P><B>Abstract</B></P> <P>The Biohydrogen Potential (BHP) of six different types of waste biomass typical for the Campania Region (Italy) was investigated. Anaerobic sludge pre-treated with the specific methanogenic inhibitor sodium 2-bromoethanesulfonic acid (BESA) was used as seed inoculum. The BESA pre-treatment yielded the highest BHP in BHP tests carried out with pre-treated anaerobic sludge using potato and pumpkin waste as the substrates, in comparison with aeration or heat shock pre-treatment. The BHP tests carried out with different complex waste biomass showed average BHP values in a decreasing order from potato and pumpkin wastes (171.1?±?7.3?ml H<SUB>2</SUB>/g VS) to buffalo manure (135.6?±?4.1?ml H<SUB>2</SUB>/g VS), dried blood (slaughter house waste, 87.6?±?4.1?ml H<SUB>2</SUB>/g VS), fennel waste (58.1?±?29.8?ml H<SUB>2</SUB>/g VS), olive pomace (54.9?±?5.4?ml H<SUB>2</SUB>/g VS) and olive mill wastewater (46.0?±?15.6?ml H<SUB>2</SUB>/g VS). The digestate was analyzed for major soluble metabolites to elucidate the different biochemical pathways in the BHP tests. These showed the H<SUB>2</SUB> was produced via mixed type fermentation pathways.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The BHP of agro-industrial waste biomass from Campania Region (Italy) is evaluated. </LI> <LI> Agro-industrial wastes are promising substrates for DF biohydrogenation. </LI> <LI> BESA effectively prepares the hydrogen producing seed inoculum. </LI> <LI> Fermentation pathways are helpful to optimize the BHP performance. </LI> </UL> </P>