Using water-sediment microcosms and 16S rRNA and 18S rRNA sequencing analyses, phenanthrene (PHE) was selected as a representative compound of PAHs to explore its effects on water quality, as well as on the diversity, structure, and function of sediment microbial communities. The results indicated that PHE pollution affected both the magnitude and stability of water quality parameters. PHE was predominantly adsorbed onto the sediment, with a degradation efficiency of 99% observed on day 56 in the PHE treatment group. Significant differences were found in bacterial communities (R_ANOSIM = 0.293, P = 0.002) and fungal communities (R_ANOSIM = 0.147, P = 0.031) among different treatment groups. The relative abundance of many bacteria, archaea, and fungi associated with PHE degradation was higher in the PHE treatment groups than that in the control group. Compared with the control and low-dosage (PHE(L)) groups, microbial networks in the medium-dosage (PHE(M)) and high-dosage (PHE(H)) groups exhibited higher average clustering coefficients and positive correlations. Functional processes such as carbon fixation, nitrogen metabolism, and sulfur metabolism had lower relative abundance in the later incubation stages in the PHE(M) and PHE(H) groups, while methane metabolism had higher relative abundance. pH was identified as the key factor influencing the structures and functions of bacterial, archaeal, and fungal communities, followed by NH₄⁺-N.