The effects of three protoporphyrinogen oxidase inhibitor herbicides, azafenidin, flurnioxazin, and sulfentrazone, on Pythium mot rot of sugarcane and the soil microbial community were evaluated in greenhouse experiments. Herbicides were applied as foliar and soil treatments. There were no consistent effects on plant growth or disease parameters. However, some herbicide treatments affected the relative frequency of isolation of Pythium spp. from roots and reduced colonization by the pathogenic species Pythium arrhenomanes. A comparison of sole carbon source utilization profiles indicated that soil-applied herbicides altered the functional diversity of the soil microbial community, with some variation depending on herbicide used. All three herbicides inhibited the in vitro mycelial growth of P. arrhenomanes, P. aphanidermatum, and P. ultimum. Active ingredients were less inhibitory than formulated product for azafenidin and flumioxazin but not for sulfentrazone.