Periphyton, comprising a group of bacteria, algae, fungi, and protozoa that grow on the surface of various substrates submerged in water, plays a crucial role in regulating nutrient cycles, such as nitrogen and phosphorus. Determining the microbial assembly process and origin of paddy periphyton, thus, could help understand the function of periphyton in nutrient cycling. In the present study, we collected soil, overlying water, and periphyton samples from paddy fields in a small subtropical catchment in Tuojia, Southern China, to analyze the soil and microbial contribution to the source of periphyton and its assembly mechanisms using amplicon sequencing, SourceTracker, and iCAMP (Infer Community Assembly Mechanisms by Phylogenetic-bin-based null model analysis). Our data showed that periphyton were widely distributed in paddy fields, with lower bacterial alpha diversity and higher eukaryotic diversity than those in the soil, and the periphyton had abundant Cyanobacteria (13.5-33.5 %), Chlorophyta (23.3-90.0 %), Bacillariophyta (5.4-40.9 %), and Nematoda (1.3-17.3 %). Overlying water was an important source of periphyton eukaryotes than that of soil (63.7 % vs. 10.0 %); however, both overlying water and soil contributed equally to the periphyton bacterial sources (38.4 % vs. 41.6 %). The periphyton community assembly was primarily driven by homogeneous selection, dispersal limitation, and drift. Homogeneous selection (50.9 %) was more significant in shaping the eukaryotic community, whereas stochastic processes (i.e., dispersal limitation and drift; 61.5 %) were more dominant in shaping the periphyton bacterial community. Our data suggest eukaryotic and bacterial community in periphyton may be controlled by two main abiotic factors: soil ammonium (NH4+-N) and Olsen phosphorus (Olsen-P). Our results reveal the potential contributions of paddy soil, overlying water, and ecological processes in shaping periphyton microbial communities in paddy fields.