The effect of steam thermotherapy on Botrytis spp. populations in strawberry transplants was evaluated. Tray plants rooted in 0.2 L peat plugs of seasonal flowering cvs. Falco, Sonsation, and Soprano, and everbearing cvs. Favori and Murano were pre-treated with steam at 37 °C for 1 h, followed by 1 h at ambient temperature and air humidity, and then 2 or 4 h steam treatment at 44 °C. Except for one cultivar with a slight reduction in yield, there were no negative effects on plant performance. Compared to untreated transplants, mean incidence of Botrytis on the five cultivars was reduced by 43 and 86% with the 2 and 4 h treatments, respectively. Within cultivars the reduction was significant in 2 and 3 experiments following the 2 and 4 h treatments, respectively. Sclerotia from four different isolates of Botrytis were subjected to treatment including 4 h of steam thermotherapy and subsequently tested for viability. Following 14 days of incubation, 90 to 100% (mean 97%) of treated sclerotia failed to produce mycelial growth compared with untreated sclerotia, which all germinated and produced mycelia. Botrytis isolates recovered from both treated and untreated strawberry transplants were tested for resistance to seven fungicides, including boscalid, fenhexamid, fludioxonil, fluopyram, pyraclostrobin, pyrimethanil and thiophanate-methyl. Multiple fungicide resistance was common; 35.5% of isolates were resistant to fungicides from at least three FRAC groups. Results indicate that steam thermotherapy treatment strongly reduces populations of Botrytis spp., including fungicide-resistant strains, in strawberry transplants with negligible negative impacts on the transplants.