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This research examined the influence of nitrogen, acetate and propionate on hydrogen production from pineapple waste extract by photosynthetic bacteria strain Rhodospirillum rubrum in batch culture. The fermentation conditions used in this study were continuous illumination fermentation (24 hours of light) and periodic illumination fermentation (alternate 12 hours of light and dark). Two levels of total nitrogen (3 mM-low level and 11 mM-high level) with various initial concentrations of acetate or propionate (5, 10 and 20 mM) were added into the production medium. Results indicated that levels of total nitrogen did not affect the production of hydrogen. Neither acetate nor propionate was used as carbon source by R. rubrum but glucose contained in pineapple waste extract was used. Periodic illuminated fermentation was more effective in producing hydrogen than continuous illuminated fermentation. The maximum hydrogen production potential (337 ml), specific hydrogen production rate (11 ml/l/h), specific hydrogen production potential (247.75 ml H 2 /g COD) and hydrogen yield (122 ml H 2 /g glucose consumed) occurred upon addition of high level of total nitrogen (11 mM) with 5 mM initial concentrations of acetate under periodic illumination and a working volume of 40 ml. Results indicated that pineapple waste extract could be effectively used as substrate for hydrogen production by R. rubrum without any carbon and nitrogen sources.