This study investigates the impact of different nutritional regimes on the bacterial communities within the root-growing substrate of a soilless tomato production system and the effects on crop quality. The experiment was conducted with two tomato varieties, Conchita and Sweetelle, under three nutritional treatments: standard, nutrient solution with 20% increased salts and nitrogen and supplementation with the biostimulant Bio-algeen S-90. Bacterial communities in the root substrate were influenced by both the tomato variety and the nutritional regime. Sweetelle exhibited more pronounced shifts in bacterial communities compared to Conchita. An overall increase in bacterial populations with time was observed (+0.38 Log). Specifically, the 20% enhanced nutrient solution had varying effects on bacterial counts in the two tomato varieties, while the biostimulant promoted an increase in ammonia-oxidizing bacteria (+0.4 Log). Microbial community analysis highlighted the distinct impact of each nutritional regime on nitrogen-cycling bacteria, which correlated with differences in quality parameters such as the L-ascorbic acid and lycopene contents. In the first case, a decrease (25–30%) was observed, while the lycopene content decreased after harvest (−51% in Conchita variety) but was more stable in the postharvest phase (66–70% lycopene retained, only 44% in the control). This study highlights how increased sources of nutrients and the differential responses of microbial communities to nutritional regimes do not necessarily increase the crop quality and that tailored approaches are required for different tomato varieties.