Milk thistle (Silybum marianum (L.) Gaertn.) is cultivated globally as a valuable medicinal plant. The presence of weeds poses numerous challenges to milk thistle production, making weed management the primary concern in milk thistle fields. Chemical weed management is an economical and promising approach to controlling weeds in cropping systems. Therefore, to investigate the tolerance of milk thistle to soil-applied herbicides, in the spring of 2022, we conducted a pot experiment as a completely randomized factorial design with four replications at the research greenhouse of the University of Birjand, Iran. The applied herbicides included metribuzin, pendimethalin, trifluralin, and ethalfluralin at six doses (0, 50, 75, 100, 125, and 150% of the recommended dose (ai ha−1)). Herbicide treatments had adverse effects on the root and shoot growth of milk thistle. Compared to the control, ethalfluralin at 150% (−60.1%) and metribuzin at 50% (−13.3%) had the highest and lowest herbicide negative effects on root dry weight, respectively. In contrast to the control, we found that ethalfluralin at 150% (−64.4%) and metribuzin at 50% (−9.3%) of the recommended dose had the highest and lowest impacts on shoot dry weight, respectively. Furthermore, herbicide applications decreased the membrane stability index (MSI) and relative water content (RWC). Root and leaf levels of malondialdehyde (MDA), total phenol, DPPH scavenging, soluble carbohydrates, and proline increased after all herbicide treatments, compared to the control. Metribuzin and pendimethalin had fewer negative effects on milk thistle growth. Consequently, these herbicides could be considered as potential options for weed control in milk thistle fields.