Protection of wounds against infection by trunk disease pathogens is the most efficient and cost-effective means to prevent grapevine trunk diseases. Studies done to determine the effectiveness of chemical pruning wound protectants have mostly focused on the control of Eutypa lata. However, other important wound pathogens, such as Phaeomoniella chlamydospora, Phaeoacremonium spp., Phomopsis spp. and species of Botryosphaeriaceae (including Botryosphaeria and aggregate genera), pose just as significant a threat to sustainable grape production. Fungicide sensitivity studies have been conducted for Pa. chlamydospora, P. viticola and E. lata. However, no such studies have been conducted for the pathogenic species of Botryosphaeriaceae from grapevines in South Africa. Ten fungicides were, therefore, tested in vitro for their efficacy on mycelial inhibition of the four most common or pathogenic species of Botryosphaeriaceae in South Africa, ‘B.’ obtusa, Neofusicoccum australe, N. parvum and Lasiodiplodia theobromae. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalid were ineffective in inhibiting the mycelial growth at the highest concentrations tested (20 µg/mL for copper ammonium acetate, 5 µg/mL for other agents tested). Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole were the most effective fungicides with EC₅₀ values for the different species ranging from 0.36–0.55, 0.07–0.17, 0.07–1.15 and 0.04–0.36 µg/mL, respectively. These fungicides, except prochloraz manganese chloride, are registered for use on grapes in South Africa and were also reported to be effective against Pa. chlamydospora, P. viticola and E. lata. Results from bioassays on 1-year-old Chenin Blanc grapevine shoots indicated that benomyl, tebuconazole and prochloraz manganese chloride were most effective in limiting lesion length in pruning wounds that were inoculated with species of Botryosphaeriaceae after fungicide treatment. The bioassay findings were, however, inconclusive due to low and varied re-isolation incidences. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can be identified as fungicides to be evaluated as pruning wound protectants in additional bioassays and vineyard trials against species of Botryosphaeriaceae as well as the other grapevine trunk disease pathogens.