This research presents the synthesis and biological evaluation of novel benzothiazole-based thiazole derivatives bearing aryl azo moieties 4a-e and 6a-e. Utilizing a one-pot three-component reaction under solvent-free conditions, benzothiazole-2-aldehyde, thiosemicarbazide, and hydrazonyl chlorides were employed to synthesize various derivatives with yields ranging from 76% to 80%. The structures of the synthesized compounds were confirmed using spectral and analytical techniques, including IR, UV-vis, and ¹H-NMR spectroscopy. Notably, the antimicrobial potential of these compounds was assessed against Gram-positive bacteria (Bacillus subtilis), Gram-negative bacteria (Escherichia coli), and fungal strains (Botrytis fabae). The derivatives demonstrated broad-spectrum antimicrobial activity, with specific compounds (e.g., 4a, 4b, 6a, and 6b) showing potency comparable to standard reference drugs like chloramphenicol and cephalothin. Structure-activity relationship analysis revealed that substitutions, such as chloro and phenyl groups, significantly influenced biological activity by enhancing lipophilicity and interaction with microbial membranes. Furthermore, molecular docking studies with PDB: 2EG7 confirmed strong binding affinities of the most active derivatives, highlighting their potential as antibacterial and antifungal agents. These findings provide valuable insights for the design of thiazole-based compounds with enhanced therapeutic properties.