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Abstract: Weed control is a key factor affecting early plant growth and establishment in revegetation projects in South-east Queensland of sub-tropical Australia. Costs associated with weed control are significant and methods which reduce establishment costs and effectively suppress weeds are of great interest. However, different methods may have implications for soil quality and fertility and require a detailed investigation. Understanding the response of soil organic matter (SOM) and microbial functional diversity to different weed control methods is crucial as they affect soil quality and nutrient availability. A field trial was established in South-east Queensland to identify the effects of three methods of weed control: (1) glyphosate, (2) a mixture of glyphosate and MCPA and (3) topsoil removal or scalping on SOM, microbial biomass, soil respiration, NH4 +-N availability, potentially mineralisable N (PMN) and soil microbial community functional diversity (as assessed by carbon substrate utilisation using Biolog GN2 plates). The scalped area had lower SOM and microbial activity compared to the herbicide and control plots. There was no significant difference in water soluble organic carbon (WSOC), hot water extractable organic C (HWEOC), hot water extractable total N (HWETN) and microbial biomass C and N (MBC and MBN) between the herbicide and control plots, particularly at week 20. NH4 +-N and PMN values were lower at week 20 than week 1 in the herbicide and scalping treatments. Week 20 was the end of the growing season and reduction in N availability may have been the result of decreasing temperature. Principal component analyses (PCA) from Biolog GN2 results indicated a separation in soil microbial community function in the scalped area compared to the other treatments which may have implications for soil properties in the long term. Scalping proved to be the most cost-effective method of site preparation, requiring fewer site visits for weed control compared to herbicide application. However, SOM was significantly affected by scalping due to topsoil removal and it may not be a sustainable practice in short rotation plantation establishment. Single herbicide application at field rates did not impact soil organisms but also failed to achieve proper weed control. [Copyright &y& Elsevier]