Heterosis, or hybrid vigor, refers to the superior phenotypes of hybrids relative to their parent inbred lines. Hybrid vigor was discovered in maize over a century ago and since then, hybrid lines comprise nearly all commercially grown grains. While quantitative genetic and molecular mechanisms have explained some heterotic patterns, the mechanism is still largely unknown. Recently, soil microbes were identified as a driver of heterosis in maize. While manipulation of the soil microbial community consistently altered heterosis, the direction of this effect appeared to be dependent on the microbiome composition, environment, or both. Here we demonstrate that both the abiotic and biotic soil environment contributes to the strength of heterosis in maize. To determine the contribution of abiotic and biotic soil factors, two inbred lines (B73 and Mo17) and their hybrid (B73xMo17) were grown in a growth chamber with four “live” and one “killed” soil treatment. The live soil treatments consisted of soils collected from two agricultural and two prairie field sites, whereas the killed treatment consisted of all soils homogenized and autoclaved. The microbial community composition, soil properties, and plant phenotypes were then assessed. After one month of growth, the reduction of soil microbes via soil sterilization resulted in increased heterosis relative to the live soil treatments. These results suggest that the absence of soil microbes impairs the growth of B73 and Mo17 but not B73xMo17 and further supports soil microbes as drivers of maize heterosis.