One-time ultrasonication pre-treatment of Rhodobacter sphaeroides was evaluated for improving biohydrogen production via photofermentation. Batch experiments were performed by varying ultrasonication amplitude (15, 30, and 45%) and duration (5, 10, and 15 min) using combined effluents from palm oil as well as pulp and paper mill as a single substrate. Experimental data showed that ultrasonication at amplitude 30% for 10 min (256.33 J/mL) achieved the highest biohydrogen yield of 9.982 mL H2/mLmedium with 5.125% of light efficiency. A maximum CODtotal removal of 44.7% was also obtained. However, when higher ultrasonication energy inputs (>256.33 J/mL) were transmitted to the cells, biohydrogen production did not improve further. In fact, 20.6% decrease of biohydrogen yield (as compared to the highest biohydrogen yield) was observed using the most intense ultrasonicated inoculum (472.59 J/mL). Field emission scanning electron microscope images revealed the occurrence of cell damages and biomass losses if ultrasonication at 472.59 J/mL was used. The present results suggested that moderate ultrasonication pre-treatment was an effective technique to improve biohydrogen production performances of R. sphaeroides.