Pendimethalin [N-(1-ethylpropyl)-3,4-dimethyl-2, 6-dinitrobenzenamine] is a herbicide used worldwide to control most annual grasses and common weeds in cereals, fruit, and vegetables. Its degradation in Haplic Chernozem under controlled greenhouse conditions was studied in this paper. The effect of recommended and doubled pendimethalin doses, as well as the effect of the biopreparate EM-EKO ProBio Plus on pendimethalin degradation in soil and on soil microorganisms was investigated. Pendimethalin half-life ranged from 24.4 to 34.4 days and the double dose did not increase the pendimethalin half-life. Thirty-eight days after pendimethalin application there was no statistical difference between the pendimethalin concentration in soil when applied at the recommended and doubled dose. No effect of pendimethalin on the amount or the activity of soil microorganisms was observed. The effect of EM-EKO ProBio Plus was apparent only on the first sampling of double-dose pendimethalin, however, this bio-preparate had no significant effect on the half-life of pendimethalin, as observed at the end of the experiment.