Effective activation of sperm motility is fundamental to successful artificial fertilisation; however, studies investigating optimal procedures in amphibians are lacking. This study found the optimal osmolality of activation media for sperm motility activation and evaluated the effect of phosphodiesterase (PDE) inhibitors on sperm activation and longevity in the critically endangered booroolong frog, Litoria booroolongensis. To assess the effect of medium osmolality (10, 25, 50, 75, 100 and 200 mOsmol kg−1) and PDE inhibitors (control, 2.5 mM caffeine, 5 mM caffeine, 2.5 mM pentoxifylline, 5 mM pentoxifylline, 2.5 mM theophylline and 5 mM theophylline) on initial activation, percentage sperm motility and sperm velocity were quantified using computer-assisted sperm analysis. To assess the effect of PDE inhibitors (control, 2.5 mM caffeine and 2.5 mM theophylline) on sperm longevity, percentage motility and velocity were assessed hourly until 10 h after activation. High (>60%) percentage motility was achieved in a broad range of activation-medium osmolalities (10–75 mOsmol kg−1). PDE inhibitors did not have an effect on initial sperm motility or velocity, but caffeine and theophylline improved sperm longevity, significantly increasing motility and velocity at 8, 9 and 10 h after activation. Data also show that sperm longevity in L. booroolongensis is extreme, with spermatozoa remaining motile more than twice as long as those of any other anuran amphibian.