The limited availability of herbicides with new mechanisms of action is remarkable given the wide range of molecular targets in plants. Allelochemicals represent promising alternatives due to their structural diversity and broader chemical spectrum compared to conventional compounds. As an initial step in exploring new herbicide mechanisms, dose-response and IC50 assays are essential to determine effective concentrations. This petri dish bioassay study evaluated the allelochemical custonolide through dose-response curves and IC50 determination on Arabidopsis thaliana (L.) Heynh. Custonolide was tested at 50, 100, 200, and 300 μM in agar, assessing germination, shoot and root lengths, and fresh and dry masses. Data were analyzed by quadratic regression in R software (expdes.pt package), and IC50 values were calculated. Results revealed a clear dose-dependent inhibition of germination and growth. Germination decreased significantly at concentrations above 200 μM, while 50 and 100 μM caused a 49.89 % reduction compared to the control, with an IC50 of 104.08 μM. Shoot and root lengths showed reductions of 49.8 % and 51.21 %, with IC50 values of 104.08 μM and 112.07 μM, respectively. Biomass was especially sensitive, with IC50 values of 3.12 μM (fresh mass) and 1.67 μM (dry mass). These results demonstrated the potent herbicidal activity of custonolide, supporting its potential as a candidate for novel herbicide development.