To clarify the relative importance of historical and contemporary environmental factors on the spatial distribution pattern of microorganisms in a river basin, this study systematically established 76 sample sites within Lancang River. From each site, five samples from each of terrestrial soil and aquatic sediment were collected. Samples sprinkling and single spore isolation methods were used to isolate Nematode-Trapping Fungi (NTF) from the samples, followed by identification of NTF using morphological and molecular biology methods. The upper, middle, and lower reaches of the Lancang River were divided by equidistance and the intersection point where the ancient Jinsha River into the ancient Lancang River as the boundary to explore the relative influence of historical factors and contemporary environmental factors on the spatial distribution pattern of NTF. A total of 566 NTF representing 31 species and 3 genera were isolated from 760 samples, with a total detection rate and Shannon Werner index of 49.74% and 2.54, respectively. Neither the detection rate nor species richness showed significant correlations with environmental factors. However, there was a significant distance decay relationship (P < 0.005) in the similarity of NTF within the basin. The NTF distribution patterns were similar under the two division models. The NTF in the upper reaches and the lower reaches had significant correlation with several environmental factors, but no significant correlation in the middle reaches (P > 0.05). NTF in the middle reaches showed significant distance attenuation under historical division (P < 0.05). The species similarity between the middle reaches of Lancang River and the upper reaches of the Jinsha River was higher (0.57 and 0.59, respectively), while it was lower (both 0.46) between their upper reaches. The results showed that there was a spatial distribution pattern of NTF in the Lancang River basin, mixed flow history of ancient Jinsha River and ancient Lancang River (historical factors) were the key driving factors, and equidistant division of basins may obscure the impact of historical factors on the spatial distribution pattern of microorganisms in the basin, this pattern can be more easily discovered by considering the evolutionary history of rivers.