Dieback caused by the fungus Lasiodiplodia theobromae is a major issue in mango plantations in the United Arab Emirates (UAE). This research discovered 36 actinobacterial isolates from mango roots in the UAE, comprising 24 classified as streptomycetes (SA) and 12 as non-streptomycetes (NSA). Eleven isolates (8 SA and 4 NSA) shown antagonistic action against L. theobromae, which is ascribed to the production of diffusible antifungal metabolites, extracellular cell-wall-degrading enzymes (CWDEs), or a combination of both. Utilizing a "novel" mango fruit bioassay, all isolates were assessed in vivo for their ability to reduce lesion severity on fruits infected with L. theobromae. Three isolates, two derived from Streptomyces and one from Actinoplanes spp., exhibited the most significant inhibitory activity against this pathogen in vitro and were subsequently selected for testing on mango seedlings. Our findings demonstrated that the antifungal efficacy of Streptomyces UAE1 was associated with antibiosis, alongside the production of CWDEs (specifically, chitinase) and siderophores; in contrast, Streptomyces UAE2 and Actinoplanes UAE1 were recognized for their respective connections to antibiotic and CWDE production. Pre-inoculation in greenhouse tests with the most promising actinobacterial isolates resulted in substantial disease protection for mango seedlings subsequently inoculated with the pathogen. The notable reduction in the projected disease severity indices of mango dieback after the application of individual biocontrol agents (BCAs), compared to the pathogen alone, confirms their effectiveness in controlling mango dieback disease. Mango seedlings infected with L. theobromae and treated with Streptomyces UAE1 shown a significant reduction in the number of defoliated leaves and conidia counts of L. theobromae, dropping by 3-fold and 6-fold, respectively, in comparison to the other two biocontrol agent treatments. This indicates that the synergistic antifungal characteristics of Streptomyces UAE1, utilizing multiple routes, hindered the in planta invasion of L. theobromae. This study is the first investigation into the effects of endophytic biological control agents against L. theobromae in mango seedlings, employing endophytic microbial antagonists. This is the first report on endophytic actinobacteria, either indigenous to the UAE or found overseas, exhibiting the ability to suppress mango dieback disease.