Excessive and inappropriate use of herbicides in crop protection can lead to soil contamination and thus to changes in this environment, which involves a serious ecological problem. A pot experiment was, a study was conducted to determine how individual and combined application of sulcotrione and terbuthylazine affect the population, diversity and structure of bacterial and fungal communities, soil enzyme activity, as well as growth and development of Zea mays L. These parameters were evaluated during a plant pot experiment conducted in a soil classified to the Eutric Cambisols type. Amending the soil with 0.115 mg kg−1 sulcotrione and 0.165 mg kg−1 terbuthylazine (whether separately or in tandem) did not significantly impact biological activity in the soil. Only the application of sulcotrione in the amount of 1.15 mg kg−1 and terbuthylazine in the amount of 1.65 mg kg−1 disturbed the microbiological and biochemical balance of the soil. Both sulcotrione alone and the sulcotrione-terbuthylazine mixture reduced the number of organotrophic bacteria and actinobacteria, whereas terbuthylazine alone increased the number of fungi. The two chemicals boosted the activity of catalase and acidic phosphatase, while also reducing the dry matter yields of maize. Dehydrogenases activity decreased under the mixture of sulcotrione-terbuthylazine. Terbuthylazine and the sulcotrione-terbuthylazine mixture additionally decreased the SPAD greenness index in maize leaves. The chemicals also affected values of the microbial colony development index and the ecophysiological diversity index. There were also shifts in the structure of bacteria from phyla Actinobacteriota and Proteobacteria, as well as fungi from phyla Ascomycota and Mortiellomycota, though the extent of these changes varied across different soils samples. It can be concluded that microbiological and enzymatic properties of soil are suitable indicators used in diagnosing the quality of soil ecosystems. Bacteria and fungi identified in soil contaminated with sulcotrione and terbuthylazine may have high potential in inactivating these chemicals. The conducted research allowed us to assess the functioning of the soil environment under the influence of single and combined use of sulcotrione and terbuthylazine. They filled the gap regarding the impact of single and combined use of sulcotrione and terbuthylazine on the number and structure of bacterial and fungal communities, enzyme activity and the growth and development of Zea mays L.