Nicosulfuron belongs to the sulfonylurea pesticides, which are widely used for weeds control. Except of benefits in plant production, long-term application of nicosulfuron may have toxic effect for living organisms, including microorganisms. The aim of this paper was to determined impact of nicosulfuron on microbial diversity of soil. Sampling of soil (0-20 and 20-40 cm) treated with nicosulfuron at village Trenica (Novi Travnik municipality, Bosnia and Herzegovina) was performed in autumn 2017. Determination of microbial diversity (total number of bacteria, ammonification bacteria, fungi and actinomycetes) was performed using standard methodology, whilst nicosulfuron-tolerant bacteria were isolated using enrichment method. Soil without nicosulfuron application was used as a control. The results showed that bacteria were most abundant microbial population. In all experiments, reduction of microbial diversity in nicosulfuron-treated soil compared to untreated was observed. This reduction was most expressed in fungal number, which is reduced from 38 to 60% compared to control. Several nicosulfuron-tolerant isolates were isolated by enrichment method. By microscopic observation and using API test kits and APIWEB database, isolates 17cs, and 22wl and 5 wl were identified as Pseudomonas fluorescens, and Bacillus subtilis, respectively. These bacterial isolates could be applied in remediation of environments polluted by nicosulfuron.