Improvements in root-zone conditions can potentially enhance shoot growth and are especially important in controlled environment systems, where maximizing yield within limited production space is essential. Commercial seaweed extract (SW) and humic acid-based biostimulants (HA) are widely used to improve root function and nutrient uptake; however, information regarding their effects through root-zone application in spinach (Spinacia oleracea L.), particularly commercial humic acid-based formulations containing microbial-derived extracts, remains limited. This study evaluated the effects of root-zone application of the commercial SW and HA products (Kelpak® and Nutra Need™, respectively) on growth, phytochemical attributes, and mineral accumulation of three spinach cultivars (‘Lakeside’, ‘Mandolin’, and ‘SV2157’) grown in pots under greenhouse conditions. Treatments included a control (no biostimulant), 1% SW (3 mL·L-1), 1% HA (3 mL·L-1), and a combined SW+HA treatment (1.5 + 1.5 mL·L-1) applied through sub-irrigation. Significant interactive effects between biostimulant and cultivar were observed for shoot and root biomass and mineral content. In ‘SV2157’, all biostimulant treatments increased shoot dry weight by approximately 34% compared with the control, while SW and SW+HA increased root dry weight by approximately 47% relative to the control. These responses were associated with greater mineral accumulation, with HA increasing N, K, Fe, S, and B and SW increasing Ca, Mg, Fe, Mn, and S compared with the control. In contrast, in ‘Mandolin’, HA and SW+HA increased root dry weight by 177–268% compared with the control; however, shoot dry weight increased only under SW, while HA and SW+HA resulted in lower N and Mg accumulation relative to the control. ‘Lakeside’ exhibited no significant root growth responses, and Ca, Fe, and Mn concentrations remained consistently lower across biostimulant treatments compared with ‘SV2157’ and ‘Mandolin’. The combined SW+HA treatment promoted balanced multi-element accumulation but did not consistently result in greater biomass than individual SW and HA applications. Leaf morphological traits were primarily cultivar dependent, whereas secondary metabolites and antioxidant activity showed limited responses to biostimulant treatments. These results indicate that the effectiveness of applying biostimulants in the root zone of spinach is strongly cultivar dependent.
