Defect engineering plays a key role in determining the catalytic and optical properties of two-dimensional (2D) materials such as molybdenum disulfide (MoS2) in their practical applications in optical and photonic devices. Here, we report a direct strategy for the fabrication of wafer-scale 2D MoS2 nanofilms with tunable sulfur (S) vacancies and crystallinity by a modified solvothermal method via a polyelectrolyte-assisted annealing process. Our results demonstrate that the S vacancies in MoS2 nanofilms can induce saturable absorption (SA) in MoS2 by introducing new energy bands within the band gap of MoS2, and the crystallinity has a significant effect on the two-photon absorption (TPA) coefficient of MoS2 nanofilms. The SA responses in MoS2 will gradually dominate the nonlinear optical (NLO) behavior of MoS2 with a lower saturable intensity along with increasing the S vacancies. The TPA coefficient of the MoS2 nanofilms with increased crystallinity is improved to (4.3 ± 0.5) × 102 cm GW-1 on increasing the crystallinity of MoS2 films, over four times larger than that of their counterpart with relatively low crystallinity. Additionally, the damage threshold of MoS2 nanofilms after polyelectrolyte-assisted annealing treatment is greatly improved to ∼74.1 GW cm-2 compared to ∼32.6 GW cm-2 of their counterpart with few S vacancies and relatively low crystallinity, due to the increased crystallinity and partial oxidation of MoS2. This work sheds light on how the defects tailor the nonlinear optical properties of 2D MoS2 nanofilms and affords an effective strategy for defect engineering via a polyelectrolyte-assisted annealing process, which can be applied to other 2D transition metal dichalcogenides.