Abstract Previous studies have shown that diverse cocktails of pesticide mixtures are omnipresent in agricultural soils yet miss a clear link to the effects on the soil microbiome. In this study, we linked the occurrence of pesticides in conventional and organic agricultural soils of the SPRINT (Sustainable plant protection transition) projects’ Case Study Sites to the composition and function of soil microbial communities. Metagenomic sequencing, phospholipid fatty acids analysis and enzyme activity measurements were used to characterize the soil microbiome and effects of site-specific parameters such as pH and SOC, and pesticide residues. Differences in the soil microbiome were strongly influenced by the geographic origin of the samples, with the pH value as dominant driver. Against our hypothesis, effects of the investigated management systems were limited, yet significant at the European level. Notably, an association between some pesticides could still be observed after accounting for the variation explained by the environmental factors. Among these, especially fungicides, with modes of action that aim to interfere with processes in microorganisms, seemed to affect the soil microbiome. This might occur either by directly affecting these processes in non-target organisms or by changing co-dependencies between fungi and bacteria. Next to fungicides, aminomethylphosphonic acid showed a significant effect on the soil microbial composition and an interactive, possibly synergistic effect with the persistent pesticide hexachlorobenzene. The latter raises concerns about a possible interaction of recently applied pesticides and persistent “legacy” pesticides. This work highlights that even though environmental parameters can overshadow the effects of pesticides, especially the identity of the pesticides present, can have an influence on the soil microbiome.