Amaranthus retroflexus L. is a common annual weed worldwide. It can be found in a wide range of habitats and causes substantial yield reduction in many crops mainly through competition. Alternaria spp. are airborne molds that are considered to have potential for the biological control of weeds. The aim of this study was to assess the effect of spore concentration, host-plant growth stage, dew period, and temperature on the pathogenicity of three Alternaria alternata isolates against A. retroflexus. The pathogenicity of A. alternata increased with increasing spore concentration and length of dew period. A spore concentration of 107 spores ml−1 in a rapeseed oil emulsion and given a 24 h dew period caused 100% mortality of A. retroflexus plants at the four-leaf stage. Infection and mortality in older plants (>four-leaf stage) was lower. The highest levels of plant mortality were obtained at post-inoculum temperatures between 20 and 30 C. These experiments confirm the potential of A. alternata as a mycoherbicide under specific environmental conditions.