BackgroundThe widespread use of chemical herbicides and the growing issue of weed resistance pose significant challenges in agriculture. To address these problems, there is a pressing need to develop biological herbicides based on bacterial metabolites.ResultsIn this study, we investigated the impact of the cell‐free culture filtrate (CFCF) from the ZT isolate, a bacilliform bacterium obtained from diseased wheat seeds, on the germination and seedling growth of various plant species, including wild oat, ryegrass, redroot, wheat, and chickpea. The results revealed that CFCF had a detrimental effect on the fresh and dry weight of stems and roots in most of the studied plants, except chickpeas. The CFCF was further subjected to separation into aqueous and organic phases using chloroform, followed by the division of the aqueous phase into 13 fractions using an alumina column. Notably, both the aqueous phase (20%) and all 13 fractions (ranging from 50% to 83%) displayed the ability to reduce the root length of ryegrass, a monocotyledonous weed. LC‐MS analysis identified that fractions 3 and 7, which were effective against ryegrass but not redroot, contained Cry family proteins, including Cry10 Aa, Cry4 Ba, and Cry4 Aa. Additionally, 16s rRNA gene sequencing revealed that the ZT isolate is closely related (98.27%) to Bacillus wiedmannii.ConclusionConclusively, metabolites from the ZT bacterium hold promise for monocotyledonous weed‐targeted herbicides, providing a constructive strategy to confront agricultural issues tied to chemical herbicides and weed resistance.This article is protected by copyright. All rights reserved.