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Grapevine trunk diseases (GTDs) are a major threat to the wine and grape industry. The aim of the study was to investigate the antifungal activity against <i>Neofusicoccum parvum</i>, <i>Diplodia seriata</i>, and <i>Botryosphaeria dothidea</i> of &#949;-polylysine, chitosan oligomers, their conjugates, <i>Streptomyces rochei</i> and <i>S. lavendofoliae</i> culture filtrates, and their binary mixtures with chitosan oligomers. In vitro mycelial growth inhibition tests suggest that the efficacy of these treatments, in particular those based on &#949;-polylysine and &#949;-polylysine:chitosan oligomers 1:1 <i>w</i>/<i>w</i> conjugate, against the three Botryosphaeriaceae species would be comparable to or higher than that of conventional synthetic fungicides. In the case of &#949;-polylysine, EC₉₀ values as low as 227, 26.9, and 22.5 &#181;g&#183;mL^&#8722;1 were obtained for <i>N. parvum</i>, <i>D. seriata</i>, and <i>B. dothidea</i>, respectively. Although the efficacy of the conjugate was slightly lower, with EC₉₀ values of 507.5, 580.2, and 497.4 &#181;g&#183;mL^&#8722;1, respectively, it may represent a more cost-effective option to the utilization of pure &#949;-polylysine. The proposed treatments may offer a viable and sustainable alternative for controlling GTDs.