Neonicotinoid pesticides are widely used within agroecosystems. Due to their systemic nature and high solubility, neonicotinoids are frequently recorded in soil, water, untreated plant matter and non-target organisms. Studies have demonstrated their capacity to induce invertebrate mortality, however, very little research has been conducted beyond pollinator exposure, particularly under field conditions. Typically, many neonicotinoids are applied via seed-dressings, reducing their direct contact with pollinators, but offering an unintended soil-exposure pathway. Soil biology underpins many vital functions, from regulating water and gas flow, to maintaining physical soil structure. In this study we investigated the effect of a commercial neonicotinoid pesticide (Insyst®) on the abundance, richness, and composition of both the mesofaunal and microbial communities and associated metabolome during oilseed rape (Brassica napus L.) production. Our results showed that over a single growing season, foliar application of Insyst® (250 g ha−1, 50 g ha−1 of the active ingredient, acetamiprid) had no significant effect (P > 0.05) on the measured soil biological indexes. Seasonal variation was a significantly greater driver in regulating biological communities within the soil than Insyst® application. In addition, we showed that the active ingredient (acetamiprid) was rapidly degraded by the soil microbial community (theoretical half-life = 119 days) during the summer cropping season. These results help highlight the need for realistic field studies, as agricultural pesticides are never pure, often containing surfactants, adjuvants, or emulsifiers which alter their behaviour and ecotoxicity. Understanding the biological interactions of vital soil fauna with necessary pesticide usage will enable proper risk alleviation measures to maintain soil biological and ecological health.