<p>Seaweed aquaculture requires reliable hatchery protocols to ensure a consistent supply of seedstock for cultivation. <i>Durvillaea</i> spp. (order Fucales), are large canopy-forming brown seaweeds native to the cool waters of the southern hemisphere and have been identified as a promising candidate for cultivation. However, components of the hatchery methods remain poorly characterised. This study investigated key factors influencing fertilisation success in <i>Durvillaea potatorum</i> determining: 1) whether storing sperm on ice enhances sperm motility and fertilisation success, 2) the effect of sperm concentration and source (different males) on fertilisation success and, 3) how the timing and volume of sperm addition affects fertilisation success. First, sperm kept on ice were motile for longer and had higher fertilisation rates over 180 minutes compared to sperm stored at room temperature. Second, sperm concentration strongly affected fertilisation success and the highest concentrations of 10⁶–10⁷ sperm mL⁻¹ resulted in much higher fertilisation rates compared to lower concentrations. There were also differences in fertilisation success among males suggesting that male-specific factors are important. Third, both the timing and volume of sperm addition affected fertilisation success. Generally, higher amounts of sperm added early (at 0 min) resulted in higher fertilisation compared to when higher amounts of sperm were later (at 10 minutes) reinforcing that fertilisation in <i>D. potatorum</i> is likely be constrained by sperm availability. Overall, our findings highlight four important factors that enhance fertilisation outcomes in <i>D. potatorum</i>, providing guidance for improving fertilisation protocols in the hatchery to support the development of a sustainable aquaculture industry.</p>