The exogenous applications of plant hormones and their analogues have been exploited to improve crop performance in the field. Protodioscin is a saponin whose steroidal moiety in its structure has some similarities with plant steroidal hormones brassinosteroids. To test the possibility that protodioscin acts as an agonist or antagonist of brassinosteroids or other plant growth regulator, in this work, we compared the responses of the weed species Bidens pilosa L. to treatment with protodioscin, brassinosteroids, auxins and abscisic acid (ABA). Protodioscin at concentrations ranging from 48 to 240 μM inhibited the growth of B. pilosa seedlings. The steroidal hormone 24-epibrassinolide (0.1 to 5 μM) also inhibited the growth of primary roots, but brassicasterol was inactive. Indole-3-acetic acid (IAA) at higher concentrations (0.5 to 10 μM) strongly inhibited the primary root length and the fresh weight of stems. ABA inhibited all parameters of seedling growth and also inhibited seed germination. The respiratory activity of primary roots (Potassium cyanide (KCN)-sensitive and KCN-insensitive respiration) was activated by protodioscin. IAA and ABA reduced the KCN-insensitive respiration. The content of malondialdehyde (MDA) in the primary roots was increased only by protodioscin treatment. All assayed compounds increased ascorbate peroxidase (APx) and peroxidase (POD) activity, with 24-epibrassinolide being much more active. The activity of catalase (CAT) was stimulated by protodioscin and 24-epibrassinolide. The overall results revealed that protodioscin was toxic to B. pilosa by a mechanism not related to plant growth regulator signalling; protodioscin caused a disturbance in mitochondrial respiratory activity, which could be related to the overproduction of ROS and consequent cell membrane damage. This article is protected by copyright. All rights reserved.