Environmentally friendly agricultural production necessitates manipulation of microbe–plant interactions, requiring a better understanding of how farming practices influence soil microbiota. We studied the effect of conventional and organic treatment on soil bacterial richness, composition, and predicted functional potential. 16S rRNA sequencing was applied to soils from adjacent plots receiving either a synthetic or organic fertilizer, where two crops were grown within treatment, homogenizing for differences in soil properties, crop, and climate. Conventional fertilizer was associated with a decrease in soil pH, an accumulation of Ag, Mn, As, Fe, Co, Cd, and Ni; and an enrichment of ammonia oxidizers and xenobiotic compound degraders (e.g., Candidatus Nitrososphaera, Nitrospira, Bacillus, Pseudomonas). Soils receiving organic fertilization were enriched in Ti (crop biostimulant), N, and C cycling bacteria (denitrifiers, e.g., Azoarcus, Anaerolinea; methylotrophs, e.g., Methylocaldum, Methanosarcina), and disease-suppression (e.g., Myxococcales). Some predicted functions, such as glutathione metabolism, were slightly, but significantly enriched after a one-time manure application, suggesting the enhancement of sulfur regulation, nitrogen-fixing, and defense of environmental stressors. The study highlights that even a single application of organic fertilization is enough to originate a rapid shift in soil prokaryotes, responding to the differential substrate availability by promoting soil health, similar to recurrent applications.