Grapevine trunk diseases (GTDs) are a significant problem for New Zealand viticulture. Endophytic actinobacteria are of interest as potential biocontrol agents due to their ability to inhibit plant pathogens and improve plant growth. However, no studies have investigated the diversity of actinobacteria associated with grapevines in New Zealand vineyards and their bioactivity. Actinobacteria diversity in different ‘Sauvignon blanc’ vine tissues from three vineyards (conventional and organic management, and different vine ages) was assessed using different methods and media. Forty-six endophytic actinobacteria were isolated, with more isolates recovered from roots (n = 45) than leaves (n = 1) and shoot internodes (n = 0). More isolates were recovered from the organic (n = 21) than conventional (n = 8) vineyard, mature (25-year old; n = 21) than young (2-year old; n = 2) vines and using a tissue maceration technique (n = 40). Actinomycete Isolation Agar, International Streptomyces Project 2, and Starch Casein media were effective for actinobacteria isolation. Most of the isolates recovered belonged to Streptomyces, with one isolate identified as Mycolicibacterium. Forty isolates were assessed for antifungal activity and plant growth-promoting (PGP) characteristics. Of these, 13 isolates had antifungal activity against test GTD pathogens (Dactylonectria macrodidyma, Eutypa lata, Ilyonectria liriodendri, Neofusicoccum parvum, and N. luteum). Eighteen isolates exhibited more than one PGP trait; 25siderophore production (n = 25), phosphate solubilization (n = 6), and indole acetic acid production (n = 16). Two strains, Streptomyces sp. LUVPK-22 and Streptomyces sp. LUVPK-30, exhibited the best antifungal and PGP properties. This study revealed the diversity of culturable endophytic actinobacteria from grapevines in New Zealand vineyards and their biocontrol potential against GTD pathogens.