In the present study, fresh olive mill waste (OMWF) was processed using an innovative pre-treatment based on the adsorption capacity of two natural matrices. The liquid fraction coming from the pre-treatment is an inexpensive feedstock for hydrogen photo-production by means of purple non-sulphur photosynthetic bacteria (PNSB). The culture broth, which contained OMWF (30%) and distilled water (70%) was tested for hydrogen photo-production using Rhodopseudomonas palustris 42OL. The photo-fermentative process was studied under batch growth conditions, at three different irradiances (18.5, 37 and 74 W/m2). The lowest cumulative amount of hydrogen (439 ml-H2/L-broth) was attained at the irradiance of 18.5 W/m2, while the highest one (1030 ml-H2/L-broth) was obtained at the irradiance of 74 W/m2. The resulting average hydrogen evolution rates (HPRavg), which were based on the culture volume and were attained at the three different irradiances (i.e. 18.5, 37 and 74 W/m2), were 1.40, 3.17 and 5.28 ml-H2/L-broth/h respectively. The culture age at which the hydrogen photo-evolution ceased showed an inverse proportionality as compared to the irradiance: i.e. the higher the irradiance, the lower the culture age. On the contrary, the COD removal efficiency (CODRE) increased depending on the irradiance. The maximal light conversion efficiency (1.07%) was attained at the irradiance of 37 W/m2, and decreased at both lower and higher irradiances. ⺠Two adsorbing vegetable matrices for olive mill waste (OMW) dephenolization. ⺠We used pre-treated OMW as feedstock for hydrogen photo-production. ⺠We set up two photo-fermentative steps to enhance the COD removal efficiency. ⺠Irradiance effects on COD removal and light conversion efficiencies were examined.