Repeated applications and combination of glyphosate-containing herbicides (GCH) with other herbicides are two weed management practices (WMP) used to compensate for GCH decreasing efficiency impacts in field crops. These practices may have serious impacts on soil functions because GCH affect soil biota and soil biological activity (SBA). Two field experiments, one with corn and one with soybean crops, were conducted during one growing season. SBA indicators, soil respiration (SR) and fluorescein diacetate (FDA) hydrolysis, were measured at two sampling times following six WMP. These WMP included one or two GCH applications (GCH alone or combined with other herbicides), applications of other herbicides only and mechanical weeding. WMP did not affect FDA neither for corn or soybean at either sampling times. In contrast, WMP affected SR in corn fields at both sampling times and SR in soybean field at the first sampling time. Repeating GCH applications and combining different herbicides led to lower SR, suggesting that these practices decreased SBA, whilst one single GCH application presented higher SR, suggesting that this practice stimulated SBA. Our study demonstrates that using GCH in combination with other herbicides or in multiple applications affects SBA in field conditions. Affecting soil functions and carbon cycle do bear serious weed management implications, and the choice of WMP should be taken into consideration to minimize their impacts on SBA for field crops sustainability.