A comparison was drawn between the effect of glyphosate (Roundup Plus), a post-emergence applied herbicide, and Harness GTZ, a pre-emergence applied herbicide, on the culturable fraction of the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize. Two different non-selective rich media were used to grow fast-growing culturable bacteria, BHI and NB, as a more accurate estimation of the soil fast-growing culturable bacterial population would be obtained from the results of cultivating in more than one medium. The potential effect was monitored by direct amplification, cloning and sequencing of bacterial DNA encoding 16S rRNA, and high-throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region from bacterial communities grown in the two different media. The estimated relative composition of the culturable maize rhizobacterial population varied considerably in accordance with the growth medium used. Both herbicides do, in fact, affect the maize rhizobacterial communities, glyphosate being, to a great extent, the less aggressive herbicide, regardless of the cultivation medium used. The pyrosequencing analysis of the fast-growing bacterial populations from the different soils represents a useful and invaluable tool to estimate the bacterial biodiversity of the culturable rhizobacteria of agricultural soils.