Actinobacteria as biological pesticides are considered an economical and safe method, especially for improving plant growth. In this study, five actinobacterial isolates were obtained from farming soil samples and evaluated for their biocontrol capacity against five or more fungal isolates that cause seedling damping-off disease in cotton. From non-sterilized cotton seeds, the frequency (%) of isolated fungi was as follows: Rhizoctonia solani (25.12%), Fusarium spp. (24.82%), and Macrophomina phaseolina (22.12%) were the most dominant fungi. Other fungi were found at lower frequencies, including Penicillium sp. (5.44%) and Aspergillus sp. (5.12%). When Koch's postulates were applied, the fungal isolates caused the same symptoms of pre- and post-emergence damping-off, as well as reduced seedling survival. R. solani, Fusarium spp., and M. phaseolina exhibited high virulence in cotton seedlings, while Penicillium sp. and Aspergillus sp. were considered moderately virulent. Several morphologically distinct actinomycete isolates were screened for chitinase production. The actinomycete isolate that produced the highest chitinase activity, indicated by the formation of a clear zone, was identified. This isolate was found to match Streptomyces gelaticus with 99.73% similarity. Antagonistic tests showed that this Streptomyces gelaticus strain inhibited all fungal pathogens. The highest inhibition zones were observed against Rhizoctonia solani, Fusarium spp., Penicillium sp., and Aspergillus sp., while Macrophomina phaseolina exhibited the lowest inhibition. In conclusion, actinomycetes, particularly Streptomyces spp., hold great potential as biocontrol agents for sustainable agriculture and the management of plant diseases.