Algal-bacterial co-cultures represent an alternative way for algal biohydrogen generation. Efficient algal hydrogen production requires anaerobiosis and electrons accessible for the algal FeFe‑hydrogenases. A number of factors strongly influence the development of this optimal environment. Various algal strains were tested for hydrogen evolution with a selected bacterial partner, a fully hydrogenase deficient Escherichia coli. During the hunt for the most efficient algae strains, gas-to-liquid phase ratio, algal optical density and algal cell size were identified as crucial factors influencing algal hydrogen evolution rate, accumulated algal hydrogen yield, carbon dioxide and oxygen levels as well as acetic acid consumption in illuminated algal-bacterial cultures. The highest accumulated hydrogen yields were observed for the different algal partners under similar experimental setup. The combination of a gas-to-liquid phase ratio of 1/1 with an algae cell density of 3.96 ∗ 108 algae cell ml−1 (OD750: 1) resulted in the highest accumulated algal hydrogen yields under continuous illumination of ~50 μmol m−2 s−1 light at 25 °C irrespective of the applied algae strain. Accumulated hydrogen yield was also strongly influenced by the algal cell size, smaller cell size correlated with higher hydrogen evolution rate. The highest accumulated algal hydrogen yield (88.98 ± 2.19 ml H2 l−1 d−1) was obtained with Chlorella sp. MACC 360 -E. coli ΔhypF co-culture.