Ethylenediaminetetraacetic acid, commonly used in micronutrient fertilizers as a chelating agent, enhances the availability of micronutrients to plants. However, its accumulation in the environment has numerous negative effects. The search for alternative chelating agents without these drawbacks is highly relevant. This study evaluated the impact of a novel micronutrient fertilizer based on the chelating agent 2-((1,2-dicarboxyethyl)amino)pentanedioic acid on the endophytic microbial community of lettuce grown in soil and hydroponic systems. The OTU count in soil was 1595, compared to 449 in hydroponics. In hydroponic systems, the application of 2-((1,2-dicarboxyethyl)amino)pentanedioic acid altered the abundance of several dominant bacterial taxa. In soil experiments, the effect of 2-((1,2-dicarboxyethyl)amino)pentanedioic acid on bacterial taxa was less pronounced. For endophytic bacterial communities, 12 bacterial OTUs were identified as core for lettuce leaves and roots regardless of the substrate. The OTU count in control soil root samples was 296, compared to 178 in hydroponic roots. The application of 2-((1,2-dicarboxyethyl)amino)pentanedioic acid led to slight shifts in the abundance of certain endophytic bacteria, especially in hydroponic systems. Non-metric multidimensional scaling analysis demonstrated that substrate type has the most significant influence on the composition of endophytic and substrate bacterial communities. The application of 2-((1,2-dicarboxyethyl)amino)pentanedioic acid did not significantly alter the composition of the communities. The results suggest that while 2-((1,2-dicarboxyethyl)amino)pentanedioic acid chelated fertilizer can influence the abundance of specific microbial taxa, it does not drastically disrupt the endophytic bacterial communities’ structure in lettuce grown in soil or hydroponics.