Assessments of incidental wildlife mortality resulting from fishing rarely account for unobserved by-catch. We assessed by-catch of protected and vulnerable wildlife species in an Australian trawl fishery by comparing in-trawl video footage with data collected by an on-board observer. Data were obtained from 44 commercial trawls with two different by-catch reduction devices (BRDs). Eighty-six individuals from six major taxa (dolphins, sharks, rays, sea snakes, turtles and sygnathids) were documented from video analysis, including the endangered scalloped hammerhead shark (Sphyrna lewini) and the critically endangered green sawfish (Pristis zijsron). On the basis of the 2008-2009 fishing effort of 4149 trawls and scaling from these results, we estimated the annual catch of protected and vulnerable species (± 1s.e.) at 8109±910 individuals. Only 34% of by-catch was expelled through the BRDs. Independent observer data for the 44 trawls showed that 77% of the landed by-catch from these taxa were dead when discarded. The results indicate that unaccounted by-catch in trawl fisheries can be substantial, and that current methods of recording by-catch on-board vessels are likely to underestimate total fishing mortality. We recommend gear modifications and their validation through dedicated observer coverage, combined with in-trawl video camera deployments to improve current approaches to by-catch mitigation. © CSIRO 2014.