Sub-Saharan Africa receives large investments in biodiversity conservation, and if these investments can be concentrated on the highest threats to biodiversity, the benefits to conservation from the investments would increase. Yet there is no available prioritization of the many direct threats to biodiversity to inform organizations developing sub-Saharan or sub-regional conservation strategies. Consequently, regional investments by funders of biodiversity conservation such as international conservation organizations, foundations, and bilateral and multilateral donors may be suboptimal. The objective of this study was to determine what are the highest direct threats to biodiversity in sub-Saharan Africa and its sub-regions. To do this, we collected threat data using standardized IUCN threat categories from a Delphi consensus of sub-Saharan Africa biodiversity experts, known threats to IUCN Red-listed sub-Saharan African species, and National Biodiversity Strategy and Action Plans from 40 sub-Saharan African countries. After averaging the threat ranking from the three sources, the highest threats were: (1) annual and perennial crops (non-timber); (2) fishing and harvesting aquatic resources in marine and freshwater areas; (3) logging and wood harvesting in natural forests; and (4) hunting and collecting terrestrial animals. The highest-ranked sub-regional threats were hunting in Central Africa and agriculture in East Africa, Southern Africa, and West Africa. Aligning biodiversity investments to address these threats and tailoring activities to reflect local socio-ecological contexts would increase the conservation of biodiversity in sub-Saharan Africa.