<p>Cassava (<i>Manihot esculenta</i> Crantz) is a crucial crop for global food security, notable for its high productivity and adaptability to low fertility soils. Micropropagation is an important tool for the multiplication and conservation of cassava genotypes, whose in vitro response depends on various factors, such as culture medium composition. In this context, understanding the effects of activated charcoal and cytokinin on the morphoanatomy of <i>M. esculenta</i> is essential to optimize propagation and conservation protocols for this species. Therefore, this study aimed to evaluate the effect of activated charcoal and cytokinin supplementation on the development and morphoanatomy of different cassava genotypes cultured in vitro. The effect of activated charcoal was assessed in an experiment conducted in a completely randomized design (CRD) in a factorial scheme (3 × 3), with three genotypes (Guatiru (BGM 0540), BRS Jari, and BRS Formosa) and three concentrations of activated charcoal (control; 0.5 and 1.0&#xa0;g L⁻¹). Subsequently, the effect of BA on micropropagation and development was investigated for the Guatiru (BGM 0540) genotype. This experiment was also conducted in a CRD with four BA concentrations (control; 0.25, 0.5, and 1&#xa0;mg L⁻¹). The presence of activated charcoal, at a concentration of 1.0&#xa0;g L⁻¹, reduced growth and differentiation of stem tissues, while promoting leaf thickening. Supplementation with BA, at a concentration of 1&#xa0;mg L⁻¹, stimulated cambial activity and increased both stem and leaf thickness in <i>M. esculenta</i>. In conclusion, both culture medium components, activated charcoal and BA, modulated the morphogenesis of <i>M. esculenta</i>, slowing plantlet development and demonstrating potential for application in in vitro propagation and conservation strategies.</p>