<p><i>Zataria multiflora</i> Boiss. is a valuable medicinal herb threatened by over-harvesting. While plant cell culture offers a sustainable alternative, a procedure for the targeted enhancement of specific phenolic classes is lacking. This study aimed to develop an optimized protocol for callus induction and suspension cultures to differentially influence metabolite accumulation in the phenylpropanoid pathway. Callus was induced from various explants on MS and B5 media. Suspension cultures were established to test the effects of hormonal composition (2,4-D, NAA, and BAP) and initial inoculum density (1, 2, and 3&#xa0;g L^− 1) on biomass and four phenolic classes. Optimal callus induction was achieved from leaf explants with 4 mg L^− 1 2,4-D + 1 mg L^− 1 BAP. In suspension cultures, a distinct hierarchical influence on metabolite accumulation was observed. Hormonal composition was the primary driver for general phenolics, with 4 mg L^− 1 2,4-D yielding the highest Total Phenolic Content (775.0&#xa0;mg GAE g^− 1 d.w.) and Total Anthocyanin Content (5.69&#xa0;mg CGE g^− 1 d.w.). Conversely, initial inoculum density was the critical modulator for specialized metabolites; a low density of 1&#xa0;g L^− 1 was optimal for Total Flavonoid Content (132.0&#xa0;mg RE g^− 1 d.w.), while 2&#xa0;g L^− 1 was best for Total Flavonol Content (168.0&#xa0;mg EE g^− 1 d.w.). High cell density (3&#xa0;g L^− 1) was strongly inhibitory to these pathways. This work establishes a robust protocol and provides novel insights into the hierarchical modulation of secondary metabolism, demonstrating that hormonal and physical parameters can be precisely manipulated for targeted biotechnological production.</p>