The study aimed to explore the biotechnological potential of actinomycetes in the synthesis of zinc oxide nanoparticles (ZnONPs) through the assessment of their ability to form a white precipitate and the detection of ultraviolet (UV) peaks. Twenty actinomycete isolates were screened, and their responses were evaluated based on the intensity of white precipitate formation (+++ for intense, + for mild, - for absent) and the presence or absence of UV peaks related to ZnONPs. The results revealed diverse behaviors among the isolates, with notable variations in both white precipitate formation and UV responses. Isolates like S13, S17, and S292 demonstrated intense white precipitate formation along with positive UV responses, suggesting a potential relationship between their ability to reduce zinc ions and ZnONP synthesis. However, isolates such as S392 and S41, despite forming intense white precipitates, displayed negative UV responses, indicating the presence of additional factors influencing ZnONP formation. These findings underscore the complexity of the biochemical pathways involved in metal ion reduction and nanoparticle synthesis by actinomycetes. The study provides valuable insights into the biotechnological applications of actinomycetes in nanomaterial synthesis and environmental remediation. Further investigations into the underlying mechanisms can enhance our understanding and pave the way for sustainable and eco-friendly technologies.