Grain legumes grown in low-rainfall (<300 mm per annum) cropping regions of southern Australia have at times failed to provide the rotational benefits observed in other regions, such as improved cereal yields in the season following a legume. ‘In-crop’ herbicides were identified as one possible factor that may have been negatively affecting the legume–rhizobia symbiosis. To test this hypothesis and identify possible mechanisms behind any observed effects, field trials were conducted at Waikerie (South Australia) in 2001, 2003, and 2004. Field pea (Pisum sativum L.) was grown and treated with one of several herbicides 5 weeks after sowing. Crop yellowing, biomass, nodulation, and nitrogen (N₂) fixation were assessed 3 weeks after spraying, and biomass, yield, percent nitrogen derived from fixation (%Ndfa), and N₂ fixation (2003, 2004) were assessed at the end of the season. Some herbicides stunted plant growth and caused crop yellowing 3 weeks after application; however, none of the herbicides affected N nutrition of peas. Despite this, in 2003, half of the herbicides assessed reduced the %Ndfa by 34–60% relative to unsprayed control plots. Herbicide effects on the measured parameters followed similar trends over each year of the 3-year study. However, effects were rarely significant in 2004 as the trials were primarily affected by low rainfall, indicating that environmental parameters play a key role in determining the severity of herbicide effects on symbiotic N₂ fixation. The possible mechanisms behind herbicide-induced damage to the pea–rhizobium symbiosis are discussed, including reduced photosynthetic capacity of plants exposed to herbicides.